Neurocyte protective agent

ABSTRACT

A protective agent for neurons of the central nervous system and a prophylactic and/or therapeutic agent for disorders in neurons of the central nervous system is provided, each including a compound represented by donepezil hydrochloride.

TECHNICAL FIELD

The present invention relates to a neurocyte protective agent comprisingdonepezil hydrochloride.

BACKGROUND ART

Donepezil hydrochloride is a substance that activates the cholinergicnervous system in the brain by reversibly inhibitingacetylcholinesterase (an enzyme which degrades acetylcholine) toincrease the amount of intracerebral acetylcholine. This substance iswidely used as a therapeutic for Alzheimer-type senile dementia andAlzheimer's disease (Japanese Patent No. 2578475), and various researchinstitutes have studied donepezil hydrochloride. Zin Zhou et al.published an article reporting that acetylcholinesterase inhibitor hasprotective effect on ischemia-like cytotoxicity of rat PC12 cells (tumorcells); in this article, donepezil hydrochloride is used as one exampleof acetylcholinesterase inhibitor (Zhou, J., Fu, Y. and Tang, X. C.,2001. Huperzine A and donepezil protect rat pheochromocytoma cellsagainst oxygen-glucose deprivation. Neurosci. Lett. 306, 53-56).

However, details of the protective effect of donepezil hydrochloride ondisorders in neurons have not been elucidated yet.

PC12 cells used in the above-mentioned article are pheochromocytomacells that are catecholamine-producing tumor derived frompheochromocytes, such as adrenal medulla or sympathetic ganglion cells.Therefore, PC12 cells are not neurons of the brain, and it is known thatPC12 cells are not forming synapse between cells and do not have afunction of responding to excitative substances (Sucher, N. J, 1993.Expression of Endogenous NMDAR1 Transcripts without Receptor ProteinSuggests Post-transcriptional Control in PC12 Cells. The journal ofBiological Chemistry. Vol. 268, No. 30, 22299-22304). That is, theabove-described article only examined cancerized cells and did not makeany examination using primary culture neurons newly prepared fromneurons of the brain. Therefore, no data have been known yet which provethe protective effect of donepezil hydrochloride on ischemia-likedisorders in actual neurons.

DISCLOSURE OF THE INVENTION

The present invention aims at providing drugs which protect neurons(especially, neurons of the central nervous system).

As a result of intensive and extensive researches toward solution of theabove problem, the present inventors have found that, surprisingly,donepezil hydrochloride has protective effect on neurons (especially,neurons of the central nervous system). Thus, the present invention hasbeen achieved based on this finding. The present invention is asdescribed below.

-   (1) A protective agent for neurons of the central nervous system,    comprising any one of the compounds shown in the following (i)    to (vii) (encompassing the compounds disclosed in Japanese    Unexamined Patent Publication No. H1-79151). The salts of these    compounds are preferably hydrochloride salts.

(i) 1-benzyl-4-[(5,6-dimethoxy-1-indanone)-2-yl]methylpiperidinerepresented by the following chemical formula or a pharmacologicallyacceptable salt thereof:

(Japanese Patent No. 2578475, Claim (hereinafter abbreviated to “cl.”)1)

(ii) a cyclic amine derivative represented by the following generalformula (I) or a pharmacologically acceptable salt thereof:

where J is a monovalent or divalent group selected from the groupsrepresented by the following formulas:

-   -   where S is a lower alkyl group with 1 to 6 carbon atoms, a lower        alkoxy group with 1 to 6 carbon groups, a halogen atom or a        hydroxyl group; t is 0 or an integer from 1 to 4; (S)_(t) may        form a methylenedioxy or ethylenedioxy group between adjacent        carbon atoms on the phenyl ring linked; Y in formula (l) is a        hydrogen atom or a lower alkyl group with 1 to 6 carbon atoms; V        in formula (k) is a hydrogen atom or a lower alkoxy group with 1        to 6 carbon atoms; W¹ and W² in formula (n) independently        represent, similarly or differently, a hydrogen atom, a lower        alkyl group with 1 to 6 carbon atoms, or a lower alkoxy group        with 1 to 6 carbon atoms; W³ is a hydrogen atom or a lower alkyl        group with 1 to 6 carbon atoms; phenyl ring A in formulas (a) to        (e), (g), (j), (l) and (q) may be substituted with an alkyl        group with 1 to 6 carbon atoms or a alkoxy group with 1 to 6        carbon atoms;    -   B is a group represented by a formula —(CHR²)_(n)— (where n is 0        or an integer from 1 to 10; R² is each independently a hydrogen        atom or a methyl group), a group represented by a formula        ═(CH—CH═CH)_(b)— (where b is an integer from 1 to 3), a group        represented by a formula ═CH—(CH₂)_(c)— (where c is 0 or an        integer from 1 to 9), or a group represented by a formula        ═(CH—CH)_(d)═ (where d is 0 or an integer from 1 to 5);    -   K is a phenylalkyl group that may have, as a substituent, an        alkyl group with 1 to 6 carbon atoms which may be halogenated,        an alkoxy group with 1 to 6 carbon atoms, a nitro group, a        halogen atom, a carboxyl group, a benzyloxy group, an        alkoxycarbonyl group with 1 to 6 carbon atoms, an amino group, a        monoalkylamino group with 1 to 6 carbon atoms, a dialkylamino        group with 1 to 6 carbon atoms, a carbamoyl group, an acylamino        group with 1 to 6 carbon atoms, a cyclohexyloxycarbonyl group,        an alkylaminocarbonyl group with 1 to 6 carbon atoms, an        alkylcarbonyloxy group with 1 to 6 carbon atoms, a hydroxyl        group, a formyl group or an alkoxy (with 1 to 6 carbon        atoms)-alkyl (with 1 to 6 carbon atoms) group; and    -   represents a single bond or a double bond;

(iii) a cyclic amine derivative selected from the compounds representedby the following formulas or a pharmacologically acceptable saltthereof:

(Japanese Patent No. 2733203, cl. 7)

(iv) a cyclic amine derivative selected from the compounds representedby the following formulas or a pharmacologically acceptable saltthereof:

(Japanese Patent No. 2733203, cl. 8)

(v) a cyclic amine derivative represented by the following generalformula (I-1) or a pharmacologically acceptable salt thereof:

-   -   where J¹⁻¹ is a lower alkyl group with 1 to 6 carbon atoms        (hereinafter, just referred to as “lower alkyl group”); a        cyclohexyl group; a phenyl, pyridyl or pyrazyl group which may        have, as a substituent, a lower alkyl group, a lower alkoxy        group with 1 to 6 carbon atoms (hereinafter, just referred to as        “lower alkoxy group”), a nitro group, a halogen, a carboxyl        group, a lower alkoxycarbonyl group, an amino group, a        mono-lower alkylamino group, a di-lower alkylamino group, a        carbamoyl group, an acylamino group derived from aliphatic        saturated monocarboxylic acid with 1 to 6 carbon atoms, a        cyclohexyloxycarbonyl group, a lower alkylaminocarbonyl group, a        lower alkylcarbonyloxy group, a halogenated lower alkyl group, a        hydroxyl group, a formyl group or a lower-alkoxy-lower-alkyl        group; a group represented by a formula        (where G is a group represented by a formula        a group represented by a formula        a group represented by a formula —O—, a group represented by a        formula        a group represented by a formula —CH₂—O—, a group represented by        a formula —CH₂—SO₂—, a group represented by a formula        or a group represented by a formula    -   E is a carbon atom or a nitrogen atom);    -   a quinolyl group; a quinoxalyl group; a furyl group or a group        represented by a formula R¹—CH═CH— (where R¹ is a hydrogen atom        or a lower alkoxycarbonyl group);    -   B is a group represented by a formula —(CH₂)_(n)—, a group        represented by a formula —NR²—(CH₂)_(n)— (where R² is a hydrogen        atom, a lower alkyl group, a phenyl group or a lower        alkylsulfonyl group), a group represented by a formula        —CONR³—(CH₂)_(n)— (where R³ is a hydrogen atom, a lower alkyl        group, a phenyl, benzyl or pyridyl group which may have, as a        substituent, a lower alkyl group, a lower alkoxy group, a        halogen or a hydroxyl group), a group represented by a formula        —NH—CO—(CH₂)_(n)—, a group represented by a formula        —CH₂—CO—NH—(CH₂)_(n)—, a group represented by a formula        —CO—CH₂—CH(OH)—CH₂—, a group represented by a formula        —CO—(CH₂)_(n)—, a group represented by a formula        —C(OH)—(CH₂)_(n)— or a group represented by a formula        —CO—CH═CH—CH₂—; and n in the above formulas is 0 or an integer        from 1 to 10;    -   T¹ is a carbon atom;    -   K is a phenylalkyl group (where the alkyl has 1 to 2 carbon        atoms) in which the phenyl may have, as a substituent, a lower        alkyl group, a lower alkoxy group, a nitro group, a halogen, a        carboxyl group, a lower alkoxycarbonyl group, an amino group, a        mono-lower alkylamino group, a di-lower alkylamino group, a        carbamoyl group, an acylamino group derived from aliphatic        saturated monocarboxylic acid with 1 to 6 carbon atoms, a        cyclohexyloxycarbonyl group, a lower alkylaminocarbonyl group, a        lower alkylcarbonyloxy group, a halogenated lower alkyl group, a        hydroxyl group, a formyl group or a lower-alkoxy-lower-alkyl        group; a cinnamyl group; a lower alkyl group; a pyridyl methyl        group; a cycloalkyl (with 3 to 6 carbon atoms)-alkyl group; an        adamantanemethyl group; a furfuryl group; a cycloalkyl group        with 3 to 6 carbon atoms; or an acyl group; and    -   q is 1 or 2; (Japanese Patent No. 3078244, cl. 1)

(vi) a cyclic amine derivative represented by the following generalformula (I-2) or a pharmacologically acceptable salt thereof:

-   -   where J¹⁻² is an indanonyl group which may have, as a        substituent, a lower alkyl group with 1 to 6 carbon atoms or a        lower alkoxy group with 1 to 6 carbon atoms; T² is a nitrogen        atom; B, K and q are the same as defined above; (Japanese Patent        No. 3078244, cl. 2)

(vii) a cyclic amine derivative selected from the compounds representedby the following formulas or a pharmacologically acceptable saltthereof:

(Japanese Patent No. 3078244, cl. 4)

-   (2) A prophylactic and/or therapeutic agent for disorders in neurons    of the central nervous system, comprising any one of the compounds    shown in the following (i) to (vii):

(i) 1-benzyl-4-[(5,6-dimethoxy-1-indanone)-2-yl]methylpiperidinerepresented by the following chemical formula or a pharmacologicallyacceptable salt thereof:

(Japanese Patent No. 2578475, cl. 1)

The salts are preferably hydrochloride salts.

(ii) a cyclic amine derivative represented by the following generalformula (I) or a pharmacologically acceptable salt thereof:

where J is a monovalent or divalent group selected from the groupsrepresented by the following formulas:

-   -   where S is a lower alkyl group with 1 to 6 carbon atoms, a lower        alkoxy group with 1 to 6 carbon groups, a halogen atom or a        hydroxyl group; t is 0 or an integer from 1 to 4; (S)_(t) may        form a methylenedioxy or ethylenedioxy group between adjacent        carbon atoms on the phenyl ring linked; Y in formula (l) is a        hydrogen atom or a lower alkyl group with 1 to 6 carbon atoms; V        in formula (k) is a hydrogen atom or a lower alkoxy group with 1        to 6 carbon atoms; W¹ and W² in formula (n) independently        represent, similarly or differently, a hydrogen atom, a lower        alkyl group with 1 to 6 carbon atoms, or a lower alkoxy group        with 1 to 6 carbon atoms; W³ in formula (n) is a hydrogen atom        or a lower alkyl group with 1 to 6 carbon atoms; phenyl ring A        in formulas (a) to (e), (g), (j), (l) and (q) may be substituted        with an alkyl group with 1 to 6 carbon atoms or a alkoxy group        with 1 to 6 carbon atoms;    -   B is a group represented by a formula —(CHR²)_(n)— (where n is 0        or an integer from 1 to 10; R² is each independently a hydrogen        atom or a methyl group), a group represented by a formula        ═(CH—CH═CH)_(b)— (where b is an integer from 1 to 3), a group        represented by a formula ═CH—(CH₂)_(c)— (where c is 0 or an        integer from 1 to 9), or a group represented by a formula        ═(CH—CH)_(d)═ (where d is 0 or an integer from 1 to 5);    -   K is a phenylalkyl group that may have, as a substituent, an        alkyl group with 1 to 6 carbon atoms which may be halogenated,        an alkoxy group with 1 to 6 carbon atoms, a nitro group, a        halogen atom, a carboxyl group, a benzyloxy group, an        alkoxycarbonyl group with 1 to 6 carbon atoms, an amino group, a        monoalkylamino group with 1 to 6 carbon atoms, a dialkylamino        group with 1 to 6 carbon atoms, a carbamoyl group, an acylamino        group with 1 to 6 carbon atoms, a cyclohexyloxycarbonyl group,        an alkylaminocarbonyl group with 1 to 6 carbon atoms, an        alkylcarbonyloxy group with 1 to 6 carbon atoms, a hydroxyl        group, a formyl group or an alkoxy (with 1 to 6 carbon        atoms)-alkyl (with 1 to 6 carbon atoms) group; and    -   represents a single bond or a double bond;

(Japanese Patent No. 2733203, cl. 1)

(iii) a cyclic amine derivative selected from the compounds representedby the following formulas or a pharmacologically acceptable saltthereof:

(Japanese Patent No. 2733203, cl. 7)

(iv) a cyclic amine derivative selected from the compounds representedby the following formulas or a pharmacologically acceptable saltthereof:

(Japanese Patent No. 2733203, cl. 8)

(v) a cyclic amine derivative represented by the following generalformula (I-1) or a pharmacologically acceptable salt thereof:

-   -   where J¹⁻¹ is a lower alkyl group with 1 to 6 carbon atoms        (hereinafter, just referred to as “lower alkyl group”); a        cyclohexyl group; a phenyl, pyridyl or pyrazyl group which may        have, as a substituent, a lower alkyl group, a lower alkoxy        group with 1 to 6 carbon atoms (hereinafter, just referred to as        “lower alkoxy group”), a nitro group, a halogen, a carboxyl        group, a lower alkoxycarbonyl group, an amino group, a        mono-lower alkylamino group, a di-lower alkylamino group, a        carbamoyl group, an acylamino group derived from aliphatic        saturated monocarboxylic acid with 1 to 6 carbon atoms, a        cyclohexyloxycarbonyl group, a lower alkylaminocarbonyl group, a        lower alkylcarbonyloxy group, a halogenated lower alkyl group, a        hydroxyl group, a formyl group or a lower-alkoxy-lower-alkyl        group; a group represented by a formula        (where G is a group represented by a formula        a group represented by a formula        a group represented by a formula —O—, a group represented by a        formula        a group represented by a formula —CH₂—O—, a group represented by        a formula —CH₂—SO₂—, a group represented by a formula        or a group represented by a formula    -   E is a carbon atom or a nitrogen atom);    -   a quinolyl group; a quinoxalyl group; a furyl group or a group        represented by a formula R¹—CH═CH— (where R¹ is a hydrogen atom        or a lower alkoxycarbonyl group);    -   B is a group represented by a formula —(CH₂)_(n)—, a group        represented by a formula —NR²—(CH₂)_(n)— (where R² is a hydrogen        atom, a lower alkyl group, a phenyl group or a lower        alkylsulfonyl group), a group represented by a formula        —CONR³—(CH₂)_(n)— (where R³ is a hydrogen atom, a lower alkyl        group, a phenyl, benzyl or pyridyl group which may have, as a        substituent, a lower alkyl group, a lower alkoxy group, a        halogen or a hydroxyl group), a group represented by a formula        —NH—CO—(CH₂)_(n)—, a group represented by a formula        —CH₂—CO—NH—(CH₂)_(n)—, a group represented by a formula        —CO—CH₂—CH(OH)—CH₂—, a group represented by a formula        —CO—(CH₂)_(n)—, a group represented by a formula        —C(OH)—(CH₂)_(n)— or a group represented by a formula        —CO—CH═CH—CH₂—; and n in the above formulas is 0 or an integer        from 1 to 10;    -   T¹ is a carbon atom;    -   K is a phenylalkyl group (where the alkyl has 1 to 2 carbon        atoms) in which the phenyl may have, as a substituent, a lower        alkyl group, a lower alkoxy group, a nitro group, a halogen, a        carboxyl group, a lower alkoxycarbonyl group, an amino group, a        mono-lower alkylamino group, a di-lower alkylamino group, a        carbamoyl group, an acylamino group derived from aliphatic        saturated monocarboxylic acid with 1 to 6 carbon atoms, a        cyclohexyloxycarbonyl group, a lower alkylaminocarbonyl group, a        lower alkylcarbonyloxy group, a halogenated lower alkyl group, a        hydroxyl group, a formyl group or a lower-alkoxy-lower-alkyl        group; a cinnamyl group; a lower alkyl group; a pyridyl methyl        group; a cycloalkyl (with 3 to 6 carbon atoms)-alkyl group; an        adamantanemethyl group; a furfuryl group; a cycloalkyl group        with 3 to 6 carbon atoms; or an acyl group; and    -   q is 1 or 2; (Japanese Patent No. 3078244, cl. 1)

(vi) a cyclic amine derivative represented by the following generalformula (I-2) or a pharmacologically acceptable salt thereof:

-   -   where J¹⁻² is an indanonyl group which may have, as a        substituent, a lower alkyl group with 1 to 6 carbon atoms or a        lower alkoxy group with 1 to 6 carbon atoms; T² is a nitrogen        atom; B, K and q are the same as defined above; (Japanese Patent        No. 3078244, cl. 2)

(vii) a cyclic amine derivative selected from the compounds representedby the following formulas or a pharmacologically acceptable saltthereof:

(Japanese Patent No. 3078244, cl. 4)

In (1) and (2) described above, the salts of the compounds arepreferably hydrochloride salts, in particular, donepezil hydrochloriderepresented by the following formula.

In (2) above, the disorders in neurons include those disorders inducedby cerebral ischemia, excitotoxicity or Aβ toxicity. As the cerebralischemia or excitotoxicity, those associated with any one of cerebralapoplexy, cerebral infarction or cerebral embolism may be given. The Aβtoxicity may be associated with Alzheimer's disease or Down's syndrome.

In (1) or (2) above, the cells are brain-derived, mature neurons; inparticular, neurons derived from one selected from the group consistingof cerebral cortex, septal area and hippocampus may be given. Theseneurons may be primary culture cells.

-   (3) A prognosis improving agent for any disease selected from    cerebral apoplexy, cerebral infarction or cerebral embolism,    comprising the protective agent described in (1) above or the    prophylactic and/or therapeutic agent described in (2) above.-   (4) A method of protecting neurons of the central nervous system,    comprising administering to a patient an effective amount of the    protective agent described in (1) above.-   (5) A method of preventing and/or treating disorders in neurons of    the central nervous system, comprising administering to a patient an    effective amount of the prophylactic and/or therapeutic agent    described in (2) above.

In (5) above, specific examples of disorders in neurons include thosedisorders induced by cerebral ischemia, excitotoxicity or Aβ toxicity.As the cerebral ischemia and excitotoxicity, those associated with anyone of cerebral apoplexy, cerebral infarction or cerebral embolism maybe given. Specific examples of excitotoxicity include one induced byNMDA or kainic acid. The Aβ toxicity may be associated with Alzheimer'sdisease or Down's syndrome.

-   (6) A method for improving the prognosis of any disease selected    from cerebral apoplexy, cerebral infarction or cerebral embolism,    comprising administering to a patient an effective amount of the    prognosis improving agent described in (3) above.-   (7) Use of any one of the compounds shown in (1) above for preparing    any agent selected from the group consisting of the protective agent    described in (1), the prophylactic and/or therapeutic agent    described in (2), and the prognosis improving agent described in    (3).-   (8) A method of screening for a compound with Aβ aggregation    inhibitory effect or a pharmacologically acceptable salt thereof,    comprising contacting cholinergic neurons of the central nervous    system with a candidate compound in the presence of Aβ and detecting    or measuring the amount of Aβ aggregation.

In the screening method of the present invention, it is possible tojudge whether or not the candidate compound has Aβ aggregationinhibitory effect by comparing the results of detection or measurementof the amount of Aβ aggregation with the amount of Aβ aggregation in theabsence of the candidate compound.

-   (9) A screening kit for a compound with Aβ aggregation inhibitory    effect or a pharmacologically acceptable salt thereof, which is for    use in the method described in (8) above.-   (10) A method for screening for a compound, or a pharmacologically    acceptable salt thereof, effective for preventing and/or treating    disorders in neurons of the central nervous system induced by Aβ    toxicity, comprising contacting cholinergic neurons of the central    nervous system with a candidate compound in the presence of Aβ and    detecting cytotoxicity or cell death.

In the method of the present invention, it is possible to judge whetheror not the candidate compound has cell protective effect against Aβtoxicity by comparing the results of detection of cytotoxicity or celldeath with the extent of cytotoxicity or cell death in the absence ofthe candidate compound.

The cytotoxicity or cell death mentioned above may be detected bymeasuring the concentration of lactate dehydrogenase (LDH) or by MTTassay.

-   (11) A screening kit for a compound, or a pharmacologically    acceptable salt thereof, effective for preventing and/or treating    disorders in neurons of the central nervous system induced by Aβ    toxicity, which is for use in the method described in (10).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing LDH release from rat primary culture cellswhen donepezil was added 12 hours and 1 hour prior to oxygen glucosedeprivation (OGD) treatment and 1 hour after OGD treatment. OGD(−)represents cells without OGD treatment, and OGD(+) represents cells withOGD treatment. “Cont” and “Vehi” are non-administration groups. The datawere analyzed by Dunnett's test. Mark “***” in FIG. 1 indicates P<0.005(“vehicle group” is used as a control).

FIG. 2 shows the results of morphological observation of rat primaryculture cells with or without OGD treatment. Panels A, B and C arephotomicrographs showing the state of the following cells; A: control;B: cells with OGD treatment; C: cells with OGD treatment after additionof donepezil 12 hours prior to the treatment. Scale bar indicates 0.1mm.

FIG. 3 is graphs showing LDH release from rat primary culture cellswhich received various acetylcholinesterase inhibitors or donepezil 12hours prior to OGD treatment. OGD(−) represents cells without OGDtreatment. OGD(+) represents cells with OGD treatment. “Cont” and “Vehi”are non-administration groups. The data were analyzed by Dunnett's test.Mark “*” in FIG. 3 indicates P<0.05; mark “**” indicates P<0.01; andmark “***” indicates P<0.005 (“vehicle group” is used as a control).

FIG. 4 is graphs showing LDH release from rat primary culture cellswhich received donepezil together with scopolamine (Sco) or mecamylamine(Mec) 12 hours prior to OGD treatment. OGD(−) represents cells withoutOGD treatment. OGD(+) represents cells with OGD treatment. “Cont” and“Vehi” are non-administration groups. The data were analyzed by ANOVAand Welch's t-test. Mark “*” in FIG. 4 indicates P<0.05; mark “**”indicates P<0.01; and mark “***” indicates P<0.005 (“vehicle group” isused as a control).

FIG. 5 is a graph showing the effect of donepezil on LDH release fromrat primary culture cells induced by NMDA treatment. Donepezil was added12 hours prior to the NMDA treatment. “Cont” and “Vehi” arenon-administration groups. The data were analyzed by Dunnett's test.Mark “***” in FIG. 5 indicates P<0.005 (“vehicle group” is used as acontrol).

FIG. 6 is a graph showing the effect of donepezil on LDH release fromrat primary culture cells induced by kainic acid treatment. Donepezilwas added 24 hours prior to the kainic acid treatment. “Cont” and “Vehi”are non-administration groups. The data were analyzed by Dunnett's test.Mark *** in FIG. 6 indicates P<0.005 (“vehicle group” is used as acontrol).

FIG. 7 shows immunostaining of cultured rat septal area neurons withanti-choline acetyltransferase antibody.

FIG. 8 is a graph showing the results of evaluation of the protectiveeffect of donepezil on Aβ(1-40) toxicity using LDH as an indicator.

FIG. 9 shows immunostaining of cultured rat septal area neurons withanti-MAP2 antibody.

FIG. 10 is graphs showing the results of measurement of Aβ aggregationusing CD changes as an indicator.

FIG. 11 is a graph showing the effect of siRNA on intracellularacetylcholine activity.

FIG. 12 is a graph showing the protective effect of siRNA on Aβ(1-40)toxicity.

FIG. 13 is graphs showing the aggregation inhibitory effect of siRNA onAβ aggregation.

BEST MODE FOR CARRYING OUT THE INVENTION

1. Outline

The present invention relates to a protective agent for neurons of thecentral nervous system and a prophylactic and/or therapeutic agent fordisorders in neurons of the central nervous system, each comprising anyone of the compounds shown below (hereinafter, referred to as the“compound Q”). According to the present invention, there are provided amethod of protecting neurons of the central nervous system and/or amethod of preventing and/or treating disorders in neurons of the centralnervous system, each characterized by administering an appropriateamount of the prophylactic and/or therapeutic agent. Hereinbelow, thecompound Q used in the protective agent, prophylactic agent, therapeuticagent and prognosis improving agent of the present invention will bedescribed.

2. Compound Q

Cyclic amine derivative represented by the following general formula (I)and pharmacologically acceptable salts thereof (see Japanese UnexaminedPatent Publication Nos. H1-79151, H07-252216 and H10-067739).

-   -   where J is:    -   (a) the following group which may be substituted or        unsubstituted: (i) a phenyl group, (ii) a pyridyl group, (iii) a        pyrazyl group, (iv) a quinolyl group, (v) a cyclohexyl        group, (vi) a quinoxalyl group or (vii) a furyl group;    -   (b) a monovalent or divalent group selected from the following        group in which the phenyl group may be substituted: (i)        indanyl, (ii) indanonyl, (iii) indenyl, (iv) indenonyl, (v)        indandionyl, (vi) tetralonyl, (vii) benzsuberonyl, (viii)        indanolyl, (ix) a group represented by a formula    -   (c) a monovalent group derived from a cyclic amide compound;    -   (d) a lower alkyl group; or    -   (e) a group represented by a formula R¹—CH═CH— (where R¹ is a        hydrogen atom or a lower alkoxycarbonyl group);    -   B is a group represented by a formula        a group represented by a formula        a group represented by a formula        where R³ is a hydrogen atom, a lower alkyl group, an acyl group,        a lower alkylsulfonyl group, a phenyl group which may be        substituted, or a benzyl group;        a group represented by a formula        where R⁴ is a hydrogen atom, a lower alkyl group, or a phenyl        group;        a group represented by a formula        a group represented by a formula        a group represented by a formula        a group represented by a formula        a group represented by a formula        a group represented by a formula        a group represented by a formula        (in the above formulas, n is 0 or an integer from 1 to 10; and        R² is a hydrogen atom or methyl group(s) in such a manner that        the alkylene group represented by a formula        does not have a substituent or has one or more methyl groups);        a group represented by a formula ═(CH—CH═CH)_(b)— (where b is an        integer from 1 to 3);        a group represented by a formula ═CH—(CH₂)_(c)— (where c is 0 or        an integer from 1 to 9);        a group represented by a formula ═(CH—CH)_(d)═ (where d is 0 or        an integer from 1 to 5);        a group represented by a formula        a group represented by a formula        a group represented by a formula        a group represented by a formula        a group represented by a formula —NH—; a group represented by a        formula —O—; a group represented by a formula —S—; a        dialkylaminoalkylcarbonyl group or a lower alkoxycarbonyl group;    -   T is a nitrogen atom or a carbon atom;    -   Q is a nitrogen atom, a carbon atom or a group represented by a        formula    -   K is hydrogen atom, a substituted or unsubstituted phenyl group;        an arylalkyl group in which the phenyl group may be substituted;        a cinnamyl group in which the phenyl group may be substituted; a        lower alkyl group; a pyridylmethyl group; a cycloalkylalkyl        group; an adamantanemethyl group; a furylmethyl group; a        cycloalkyl group; a lower alkoxycarbonyl group; or an acyl        group;    -   q is an integer from 1 to 3;    -   represents a single bond or a double bond.

In the above-mentioned definitions for compound (I), the lower alkylgroup in J, K, R³ and R⁴ means a straight-chained or branched alkylgroup with 1 to 6 carbon atoms, such as methyl group, ethyl group,propyl group, isopropyl group, butyl group, isobutyl group, sec-butylgroup, tert-butyl group, pentyl group (amyl group), isopentyl group,neopentyl group, tert-pentyl group, 1-methylbutyl group, 2-methylbutylgroup, 1,2-dimethylpropyl group, hexyl group, isohexyl group,1-methylpentyl group, 2-methylpentyl group, 3-methylpentyl group,1,1-dimethylbutyl group, 1,2-dimethylbutyl group, 2,2-dimethylbutylgroup, 1,3-dimethylbutyl group, 2,3-dimethylbutyl group,3,3-dimethylbutyl group, 1-ethylbutyl group, 2-ethylbutyl group,1,1,2-trimethylpropyl group, 1,2,2-trimethylpropyl group,1-ethyl-1-methylpropyl group, or 1-ethyl-2-methylpropyl group. Of thesegroups, preferable are methyl group, ethyl group, propyl group andisopropyl group. Methyl group is most preferable.

In the definition “the following group which may be substituted orunsubstituted: (i) a phenyl group, (ii) a pyridyl group, (iii) a pyrazylgroup, (iv) a quinolyl group, (v) a cyclohexyl group, (vi) a quinoxalylgroup or (vii) a furyl group” in J, specific examples of the substituentinclude lower alkyl groups with 1 to 6 carbon atoms, such as methylgroup, ethyl group, n-propyl group, isopropyl group, n-butyl group,isobutyl group, or tert-butyl group; lower alkoxy groups correspondingto the above-mentioned lower alkyl groups, such as methoxy group orethoxy group; nitro group; halogens such as chlorine, bromine orfluorine; carboxyl group; lower alkoxycarbonyl groups corresponding tothe above-mentioned lower alkoxy groups, such as methoxycarbonyl group,ethoxycarbonyl group, isopropoxycarbonyl group, n-propoxycarbonyl group,or n-butyloxycarbonyl group; amino group; mono-lower alkylamino group;di-lower alkylamino group; carbamoyl group; acylamino groups derivedfrom aliphatic saturated monocarboxylic acid with 1 to 6 carbon atoms,such as acetylamino group, propyonylamino group, butylylamino group,isobutylylamino group, valerylamino group or pivaloylamino group;cycloalkyloxycarbonyl groups such as cyclohexyloxycarbonyl group; loweralkylaminocarbonyl groups such as methylaminocarbonyl group orethylaminocarbonyl group; lower alkylcarbonyloxy groups corresponding tothe above-defined lower alkyl groups, such as methylcarbonyloxy group,ethylcarbonyloxy group or n-propylcarbonyloxy group; halogenated loweralkyl groups represented by trifluoromethyl group; hydroxyl group;formyl group; and lower-alkoxy-lower-alkyl groups such as ethoxymethylgroup, methoxymethyl group or methoxyethyl group. In the aboveexplanation of the substituent, it is intended that the “lower alkylgroup” and the “lower alkoxy group” include every group deriving fromthe above definitions. The group in J may be substituted with 1 to 3substituents which may be the same or different.

Further, in the case of phenyl group, such a group as shown below isalso included in the “substituted phenyl group” used herein:

A group represented by a formula

where G is a group represented by a formula

a group represented by a formula

a group represented by a formula —O—; a group represented by a formula

a group represented by a formula —CH₂—O—; a group represented by aformula —CH₂—SO₂—; a group represented by a formula

or a group represented by a formula

andE is a carbon atom or a nitrogen atom.

Of those substituents, preferable substituents for phenyl group include,but are not limited to, lower alkyl groups, lower alkoxy groups, nitrogroup, halogenated lower alkyl groups, lower alkoxycarbonyl groups,formyl group, hydroxyl group, lower-alkoxy-lower-alkyl groups, halogenatoms, benzoyl group and benzylsulfonyl group. The substituents may betwo or more, which may be the same or different.

Preferable substituents for pyridyl group include, but are not limitedto, lower alkyl groups, amino group and halogen atoms.

Preferable substituents for pyrazyl group include, but are not limitedto, lower alkoxycarbonyl groups, carboxyl group, acylamino group,carbamoyl group and cycloalkyloxycarbonyl group.

The pyridyl group as J is preferably 2-pyridyl group, 3-pyridyl group or4-pyridyl group; the pyrazyl group as J is preferably 2-pyrazyl group;the quinolyl group as J is preferably 2-quinolyl group or 3-quinolylgroup; the quinoxalyl group as J is preferably 2-quinoxalyl group or3-quinoxalyl group; and the furyl group as J is preferably 2-furylgroup.

In the definition of J, representative examples for groups (i) to (ix)described in (b) are as follows.

In the above-described series of formulas, t is 0 or an integer from 1to 4; and S is one of the substituents (which may be the same ordifferent) defined in (a) in J or a hydrogen atom. Preferably, S is ahydrogen atom (unsubstituted), a lower alkyl group or a lower alkoxygroup. Further, S may be substituted with an alkylenedioxy group (suchas methylenedioxy group or ethylenedioxy group) between adjacent carbonatoms on the phenyl ring.

Most preferably, S is not substituted or have 1 to 3 methoxy groupssubstituted.

The indanolydenyl shown above is one example of the divalent group inwhich the phenyl group may be substituted in the definition of (b) in J.Indanolydenyl is a representative divalent group derived from indanonylof (ii) in (b) in J.

In the definition of J, the monovalent group derived from circular amidecompounds includes, but is not limited to, quinazolone,tetrrahydroisoquinoline-one, tetrahydrobenzodiazepine-one andhexahydrobenzazocine-one. Any monovalent group having a circular amidein its structural formula may be included.

The circular amide can be derived from a single ring or a condensedhetero-ring. Preferably, the condensed hetero-ring is one condensed witha phenyl ring. In this case, the phenyl ring may be substituted withlower alkyl groups with 1 to 6 carbon atoms (preferably methyl group),lower alkoxy groups with 1 to 6 carbon atoms (preferably methoxy group)or halogen atoms.

Preferable examples are given below.

In the above formulas, Y in formulas (i) and (l) is a hydrogen atom or alower alkyl group; V in formula (k) is a hydrogen atom or a lower alkoxygroup; W¹ and W² in formulas (m) and (n) represent a hydrogen atom, alower alkyl group or a lower alkoxy group; and W³ in formula (n)represents a hydrogen atom or a lower alkyl group.

In formulas (j) and (l), the ring on the right is a seven-membered ring.In formula (k), the ring on the right is a eight-membered ring.

Among the definitions of J described above, the most preferable is amonovalent group derived from indanone in which the phenyl ring may besubstituted or a monovalent group derived from a circular amidecompound.

In the definition of B, the group represented by a formula

is represented by a formula —(CH₂)_(n)— when R² is a hydrogen atom;further, this formula means that one or more methyl groups may be linkedto any of the carbon atoms of the alkylene chain. In this case, n ispreferably 1 to 3.

In the series of formulas shown in the definition of B, a group havingan amide group is also a preferable group.

Examples of more preferable groups include a group represented by aformula ═(CH—CH═CH)_(b)— (where b is an integer from 1 to 3); a grouprepresented by a formula ═CH—(CH₂)_(c)— (where c is 0 or an integer from1 to 9); a group represented by a formula ═(CH—CH)_(d)═ (where d is 0 oran integer from 1 to 5); a group represented by a formula —NH—; a grouprepresented by a formula —O—; or a group represented by a formula —S—.

Ring

may be a five- to seven-membered ring. Specifically,

may be given. A particularly preferable ring is a piperidine ringrepresented by a formula

In the terms “substituted or unsubstituted phenyl group” and“substituted or unsubstituted arylalkyl group” used in the definition ofK, the substituent is the same as defined in (i) to (vii) in (a) in thedefinition of J.

The arylalkyl group means benzyl group, phenethyl group, etc. in whichthe phenyl group is substituted with the above-mentioned substituent ornot substituted.

Specific examples of pyridylmethyl group include 2-pyridylmethyl group,3-pyridylmethyl group and 4-pyridylmethyl group.

Most preferable K is an arylalkyl group in which the phenyl group may besubstituted, a substituted or unsubstituted phenyl group, or a cinnamylgroup in which the phenyl group may be substituted.

Specific examples of preferable arylalkyl group include benzyl group andphenethyl group in which the phenyl group may be substituted with alower alkoxy group with 1 to 6 carbon atoms, a lower alkyl group with 1to 6 carbon atoms, a hydroxyl group, etc.

represents a single bond or a double bond.

As an example where the bond is a double bond, the above-describeddivalent group derived from indanone in which the phenyl ring may besubstituted, i.e., indanolydenyl group may be given.

In the present invention, specific examples of pharmacologicallyacceptable salts include inorganic acid salts such as hydrochloridesalts, sulfate salts, hydrobromate salts and phosphorate salts; andorganic acid salts such as formate salts, acetate salts,trifluoroacetate salts, maleate salts, tartarate salts, methanesulfonatesalts, benzenesulfonate salts and toluenesulfonate salts.

Depending on the selection of substituents, the compound may form alkalimetal salts such as sodium salts and potassium salts; alkaline earthmetal salts such as calcium salts and magnesium salts; organic aminesalts such as trimethylamine salts, triethylamine salts, pyridine salts,picoline salts, dicyclohexylamine salts and N,N′-dibenzylethylenediaminesalts; and ammonium salts.

In the present invention, the above-described compound may haveasymmetric carbon atoms depending on the type of the substituent. Thus,optical isomers may exist. Needless to say, such optical isomers areincluded in the scope of the present invention.

In one specific example when J has the indanone skeleton, J hasasymmetric carbon atoms and thus geometrical isomers, optical isomers,diastereomers, etc. may exist. Such geometrical isomers, opticalisomers, diastereomers, etc. are included in the scope of the presentinvention.

Taking all the definitions into consideration, a group of compoundsespecially preferable are as described below. A cyclic amine representedby the formula (A) or a pharmacologically acceptable salt thereof:

-   -   where J¹ is a monovalent or divalent group selected from the        following group in which the phenyl group may be        substituted: (i) indanyl, (ii) indanonyl, (iii) indenyl, (iv)        indenonyl, (v) indandionyl, (vi) tetralonyl, (vii)        benzsuberonyl, (viii) indanolyl or    -   (ix) a group represented by a formula    -   B, T, Q, q and K are the same as defined above.

In the definition of J¹ described above, most preferable group is anindanonyl, indandionyl or indanolydenyl group in which the phenyl groupmay be substituted. In this case, most preferably, the phenyl group isunsubstituted or substituted with the same or different substituentsthat are hydroxyl groups, halogen atoms and lower alkoxy groups.Examples of lower alkoxy groups include methoxy group, ethoxy group,isopropoxy group, n-propoxy group and n-butoxy group each having 1 to 6carbon atoms. They may be mono- to tetra-substituted. Preferably, thelower alkoxy group is di-substituted. Most preferable is di-substitutedmethoxy group.

A group of compounds which are included in formula (A) above and arestill more preferable can be represented by the following generalformula (B).

-   -   where J¹ is a monovalent or divalent group selected from the        following group in which the phenyl group may be        substituted: (i) indanyl, (ii) indanonyl, (iii) indenyl, (iv)        indenonyl, (v) indandionyl, (vi) tetralonyl, (vii)        benzsuberonyl, (viii) indanolyl or    -   (ix) a group represented by a formula    -   B¹ is a group represented by a formula    -   where n is 0 or an integer from 1 to 10; R¹ is a hydrogen atom        or a methyl group in such a manner that the alkylene group        represented by a formula        does not have a substituent or has one or more methyl groups;    -   a group represented by a formula ═(CH—CH═CH)_(b)— (where b is an        integer from 1 to 3);    -   a group represented by a formula ═CH—(CH₂)_(c)— (where c is 0 or        an integer from 1 to 9);    -   a group represented by a formula ═(CH—CH)_(d)═ (where d is 0 or        an integer from 1 to 5);    -   T, Q, q and K are the same as defined above.

A group of compounds which are included in formula (B) above and arestill more preferable can be represented by the following generalformula (C).

where J¹, B¹ and K are the same as defined above.

This formula corresponds to a case where the group represented by theformula

is the group represented by a formula

i.e., piperidine.

A group of compounds which are included in formula (C) above and arestill more preferable can be represented by the following generalformula (D).

-   -   where J² is a group selected from an indanonyl, indandionyl or        indanolydenyl group in which the phenyl group may be        substituted;    -   K¹ is a substituted or unsubstituted phenyl group, an arylalkyl        group which may be substituted, or a cinnamyl group which may be        substituted; and    -   B¹ is the same as defined above.        3. Protection of Neurons

In the present invention, “neurons of the central nervous system” meansbrain-derived, mature neurons (preferably, human- or othermammal-derived neurons of the central nervous system which have beeninvolved in neuronal network and matured functionally); they are derivedfrom one selected from the group consisting of cerebral cortex, septalarea and hippocampus.

In the present invention, protection of neurons of the central nervoussystem means protective effect upon neurons of the central nervoussystem against various loads resulted from ischemia-like effect such ascerebral ischemia; resulted from excitotoxicity induced by excitantsubstances such as NMDA (N-methyl-D-aspartate) or kainic acid; resultedfrom peptide or protein aggregate toxicity (including Aβ toxicity andprion toxicity; resulted from NO (nitric oxide) or active oxygenspecies; and so forth. Disorders of neurons induced by cerebral ischemiainclude disorders induced by degenerative diseases, etc. of cerebralneurons such as cerebral apoplexy, cerebral infarction or cerebralembolism (which is said recently to be the cause of Alzheimer'sdisease). The above-mentioned peptide or protein aggregate toxicity(especially Aβ toxicity) includes those which are induced in associationwith Alzheimer's disease or Down's syndrome.

The above-mentioned “protection of neurons” is used in a wide meaning.For example, protection of neurons include not only to actually preventthe death of neurons of the central nervous system caused by loads onthem (e.g., physical or chemical disorders undesirable for themaintenance of homeostasis, such as stress, trophopathy, diseases,injuries, decreased strength due to surgical operations or the like,prostration, aging; or cytotoxicity) but also to prevent the lowering offunctions of neurons.

In order to examine whether neurons have been protected or not, neuronsmust be prepared. The neurons for this purpose are not particularlylimited. They may be primary culture cells prepared from biologicalsamples. With respect to preparation of primary culture cells frombiological samples, for example, cerebral cortex-derived primary cultureneurons may be prepared from cerebral cortex, and septal area-derivedprimary culture neurons may be prepared from the septal region, i.e.,the region including septal area and basal forebrain.

Animal species from which cells are collected are not particularlylimited. For example, rat, mouse, guinea pig, hamster, rabbit or thelike may be used. Although cells may be taken at any stage of growthfrom embryo to adult, it is preferable to take cells from embryos (e.g.,18-day-old embryos). Tissues taken are treated with such as trypsin orcollagenase to thereby obtain neurons.

Cell culture may be performed by conventional methods for culturinganimal cells. One of ordinary skill in the art can select appropriateculture conditions.

4. Protective Agent for Neurons and Method of Protection

Compound Q described earlier has protective effect against disorders ofneurons (especially, neurons of the central nervous system). Also, thesiRNA of acetylcholinesterase gene (AChE gene) inhibits the expressionof ACHE gene to thereby inhibit the function of ACHE, and thus inhibitsthe aggregation of Aβ. Therefore, in the present invention, compound Qor the siRNA of ACHE gene described later is useful as an activeingredient in a protective agent for neurons of the central nervoussystem against degenerative diseases of cerebral neurons such ascerebral apoplexy, cerebral infarction or cerebral embolism, and aprophylactic, therapeutic and/or prognosis improving agent for disordersof neurons (hereinafter, sometimes referred to as the “protective agent,etc. of the present invention”). A therapeutic agent, treating method,prognosis improving agent and prognosis improving method for Alzheimer'sdisease and Down's syndrome are provided by the present invention.

The active ingredient of the protective agent etc. of the presentinvention (e.g., donepezil hydrochloride) may be either an anhydride ora hydrate. Further, the above donepezil can have crystal polymorphism,for example. In this case, a single crystal form or a mixture of crystalforms may be used.

Compound Q used in the present invention (e.g., donepezil hydrochloride)may be prepared by known methods. For example, the compound can beprepared easily by the method disclosed in Japanese Unexamined PatentPublication No. H1-79151, Japanese Patent No. 2578475, Japanese PatentNo. 2733203 or Japanese Patent No. 3078244. Donepezil hydrochloride isalso available as a preparation in the form of fine granules or thelike.

The siRNA used in the present invention is designed by the methoddescribed later. One of ordinary skill in the art can prepare the siRNAby known methods using a nucleic acid synthesizer or the like.

It is possible to use compound Q or the siRNA of ACHE gene as it is inthe protective agent etc. of the present invention. Alternatively, it isalso possible to formulate the compound or the siRNA into a preparationtogether with known, pharmacologically acceptable carriers and the like.Examples of pharmacologically acceptable carriers include excipients,binders, disintegrants, lubricants, coloring agents, flavoring agents,stabilizers, emulsifiers, absorption enhancer, surfactants, pHadjusters, preservatives, antioxidants, etc. Forms of preparationsinclude tablets, powders, fine granules, granules, capsules, syrups andthe like for oral administration and suppositories, injections,ointments, poultices and the like for parenteral administration.

The route of administration of the protective agent etc. of the presentinvention is not particularly limited. The protective agent may beadministered orally or parenterally. For example, oral dosage forms ofdonepezil hydrochloride are available as Aricept™ fine granules (Eisai)and Aricept™ tablets (Eisai). With respect to forms of parenteraladministration, transdermal absorption, intravenous injection,subcutaneous injection, intradermal injection, intramuscular injection,intraperitoneal injection and the like may be enumerated. Intravenousinjection is preferable. Injections may be prepared as non-aqueousdiluents (e.g., glycols such as propylene glycol, polyethylene glycol,vegetable oils such as olive oil, alcohols such as ethanol), suspensionsor emulsions. The asepticizing of injections may be performed by filtersterilization, addition of sterilants, or the like. Alternatively,injections may take a form which is prepared at the time of use. Thatis, an aseptic solid composition is prepared by lyophilization or thelike, and this composition is dissolved in solvent such as asepticdistilled water for injections before use. When the protective agentetc. of the present invention is administered by transdermal absorptionin the form of a patch, it is preferable to select the so-called freeform which does not form salts.

The dose of compound Q in oral administration is preferably 0.1-100mg/day, more preferably 1.0-50 mg/day, taking donepezil hydrochloride asan example. Preferable dos of the siRNA of ACHE gene is 0.01-100 mg/day,more preferably 0.1-50 mg/day.

In parenteral administration, when patches are used, the dose ispreferably 5-50 mg/day, more preferably 10-20 mg/day. In the case ofinjections, they may be prepared by dissolving or suspending in apharmacologically acceptable carrier (such as physiological saline orcommercial distilled water for injections) to give a concentration of0.1 μg/ml carrier to 10 mg/ml carrier. The dose of the thus preparedinjection is 0.01-5.0 mg/day, more preferably 0.1-1.0 mg/day forpatients who need treatment. This dose may be administered at once ordivided into two or three administrations per day.

5. Method of Evaluating Performance

The effect of the protective agent of the invention on neurons againstischemic disorders can be evaluated by OGD (oxygen glucose deprivation)test. Further, the protective agent of the invention on neurons againstexcitotoxicity can be evaluated by NMDA or kainic acid stimulation test.Further, the protective agent of the invention on neurons against Aβtoxicity can be evaluated by Aβ toxicity test or Aβ aggregation test.Hereinbelow, methods of individual tests will be described.

(1) OGD Test

In OGD test, a model is used in which ischemia-like cytotoxicity isinduced in rat primary culture cerebral cortex neurons by giving a loadvia oxygen glucose deprivation. Whether donepezil hydrochloride hasprotective effect on neurons against the ischemia-like disorder or notis examined using that model.

In this Example, primary culture neurons can be prepared from thecerebral cortex of rat embryos (17-19-day-old embryos). Cells which havebeen cultured for more than 7 days under conventional culture conditionsfor animal cells (e.g., at 37° C. under 5% CO₂) may be used. OGDtreatment is performed by rat primary culture cerebral cortex neurons ina glucose-free buffer and transferring them into a tightly sealedchamber where nitrogen replacement is carried out to create a low oxygenenvironment. Cells after OGD treatment are transferred from theglucose-free buffer to a cell culture medium and cultured overnight at37° C. under 5% CO₂.

-   (A) In order to clarify whether compound Q (e.g., donepezil) has    protective effect on neurons against ischemic disorders or not,    first, this Example is performed under conditions where the compound    is added before and after the OGD treatment. For example, neurocyte    protective effects are compared among the following groups: “Pre-12    h” where donepezil was added 12 hours before OGD treatment; “Pre-1    h” where donepezil was added 1 hour before OGD treatment; “Post-1 h”    where donepezil was added 1 hour after OGD treatment; “Cont” where    no OGD treatment was given; and “Vehicle” where no donepezil was    added before or after OGD treatment. As an indicator for the effect    of neurocyte protection, ratio of LDH release inhibition can be    used. LDH (lactate dehydrogenase) is an oxidation-reduction enzyme    present in cytoplasm and converts pyruvic acid into lactic acid to    thereby reduce the amount of intracellular NADH (nicotinamide    adenine dinucleotide). Therefore, when cells are injured by OGD, LDH    flows out from inside of the cells into extracellular solution. In    this solution, LDH is present depending on the degree of injury of    the cells (cell death). The amount of LDH present in the solution    can be determined by adding pyruvic acid and NADH to the solution    and measuring the decreasing ratio of NADH with an absorption    spectrometer. According to this testing example, it is revealed that    the highest neurocyte protection effect is produced when donepezil    was added 12 hours before OGD treatment (“Pre-12 h”).-   (B) It is also possible to observe the degree of injury to the cells    or the degree of protection of neurons by the compound by    microscopically observing the morphology of the OGD treated, rat    primary culture neurons. When compared with control cells,    vehicle-treated cells do not take the normal form. On the other    hand, when donepezil was added before OGD treatment, serious injury    as seen in vehicle-treated cells is not observed and cells show a    form close to that seen before OGD treatment. Therefore, from the    viewpoint of cellular morphology, it is also demonstrated that    neurons are protected by adding donepezil before OGD treatment.-   (C) Further, another test is possible. Briefly, rat primary culture    neurons are subjected to OGD treatment in the same manner as in the    above-described test. At that time, various acetylcholinesterase    inhibitors (galantamine, tacrine and rivastigmine) and donepezil are    added to the cells in varied concentrations to examine their effect    upon LDH release. In this test, while the acetylcholinesterase 50%    inhibition concentration of donepezil is almost equivalent to that    of rivastigmine, rivastigmine and other acetylcholinesterase    inhibitors did not show any neurocyte protective effect. This    suggests that the protective effect of donepezil on OGD treated    neurons is based on a mode of action which is different from    acetylcholinesterase inhibitory action.-   (D) Subsequently, in order to examine whether the above-mentioned    protective effect of donepezil on neurons is mediated by    acetylcholine receptor or not, acetylcholine receptor antagonists    scopolamine (muscarine receptor antagonist) and mecamylamine    (nicotine receptor antagonist) are added to cells to see how the    protective effect of donepezil will be affected. According to this    test, the protective effect of donepezil on neurons is not affected    by the addition of scopolamine and mecamylamine; thus, it is    demonstrated that the effect of donepezil is not mediated by    acetylcholine receptor.    (2) Excitotoxicity Test    (A) NMDA Toxicity

In excitotoxicity test, protective effects of compounds (e.g.,donepezil) on primary culture neurons against NMDA(N-methyl-D-aspartate) toxicity are examined. A model is used in whichcytotoxicity is induced in rat primary culture cerebral cortex neuronsby NMDA stimulation. Briefly, NMDA is added (e.g., 100 μM for 9 hours)to primary culture neurons obtained as described above and then theamount of LDH in the culture medium is measured. Donepezil is added tocells before the addition of NMDA stimulation, e.g., 12 hours before theNMDA stimulation. Then, after addition of NMDA, the amount of LDH in theculture medium may be measured, for example, 9 hours after the additionand used as an indicator for neurocyte protection effect.

According to this test, it is clear that donepezil shows protectiveeffect on neurons against NMDA excitotoxicity in aconcentration-dependent manner.

(B) Kainic Acid Toxicity

It is said that kainic acid enhances the death of neurons induced byβ-amyloid which is said one of the causative factors of Alzheimer'sdisease. In this test, protective effects of compounds (e.g., donepezil)on rat primary culture cerebral cortex neurons against cytotoxicityinduced by kainic acid are examined.

Kainic acid treatment may be performed, for example, by adding kainicacid to the medium of cells which have been cultured for 7 days or moreand then culturing the cells overnight at 37° C. under 5% CO₂. CompoundQ (e.g., donepezil) may be added to the cells, for example, 24 hoursbefore the addition of kainic acid stimulation. After addition of kainicacid, the amount of LDH present in the culture medium is measured, forexample, 24 hours after the addition and used as an indicator forneurocyte protection effect. In this test, donepezil inhibits the LDHrelease of neurons in a dose-dependent manner. Therefore, it is clearthat donepezil shows protective effect against kainic acidexcitotoxicity in a concentration-dependent manner.

(3) Aβ Toxicity Test or Aβ Aggregation Test

(A) Cultured Septal Area Neurons

Aβ is considered to be a cause of Alzheimer's disease, and centralcholinergic nerves involved in memory and learning are lost inAlzheimer's disease. And cholinergic nerves are projected from septalarea to regions such as hippocampus that are susceptible to disorders inAlzheimer's disease. Taking into consideration the above-describedfindings and the fact that volume reduction in hippocampus caused byAlzheimer's disease is inhibited by administration of donepezilhydrochloride, it is preferable to analyze the protective effect againstAβ toxicity and Aβ aggregation inhibitory effect of compounds (e.g.,donepezil) in cholinergic nerves.

A great number of septal area neurons have choline acetyltransferase(ChAT) (an acetylcholine synthesis enzyme) and thus are believed to becholinergic nerves. The present invention has demonstrated for the firsttime in the above-mentioned septal area neurons (which are cholinergicnerves) that the Aβ aggregation inhibitory effect of donepezil protectscholinergic neurons against toxicity.

Septal area neurons may be prepared by taking cells from living bodiesand culturing them. They may be derived from any one of mouse, rat,guinea pig, hamster, rabbit, or the like; the species is notparticularly limited. Although cells may be taken at any stage of growthfrom embryo to adult, it is preferable to take cells from embryos (e.g.,18-day-old embryos). The septal region of the brain (i.e., the regionincluding septal area and basal forebrain) is cut out and treated withtrypsin to thereby obtain cells.

Septal area neurons may be obtained by plating the thus obtained cellson culture plates at an appropriate concentration and culturing them.Preferably, the culture plates are coated with poly-D-lysine. The cellconcentration is preferably 1.2×10⁵ cells per well of, for example,96-well poly-D-lysine-coated plates. As the medium, 5% fetal calfserum-containing DMEM may be used. Still preferably, DMEM contains 5μg/ml insulin, 30 nmol/L sodium selenite, 100 μmol/L putrescine, 20nmol/L progesterone, 15 nmol/L biotin, 100 units/ml penicillin, 100μg/ml streptomycin, 1 mmol/L sodium pyruvate, and the like.

The confirmation that cultured septal area neurons are cholinergicnerves can be made using the expression of ChAT as an indicator asdescribed above. The presence or absence of ChAT expression can beconfirmed by immnunostaining the cultured septal area neurons withanti-ChAT antibody.

(B) Detection of Aβ Aggregation

In the present invention, Aβ aggregation can be examined using as anindicator changes in CD (circular dichroism) spectra at 215-260 nmattributable to the formation of a β-sheet structure of Aβ. CD spectraat 215-260 nm decrease when Aβ forms an α-helix structure or a β-sheetstructure. In particular, it is known that the formation of a β-sheetstructure decreases CD spectra around 215 nm.

In another embodiment, fluorescence of Aβ may be measured withthioflavin T to examine Aβ aggregation in culture medium simply.Briefly, 48 hours after addition of Aβ(1-42) to cells, medium samplesare taken and 10 μmol/L of thioflavin T is added thereto. Immediatelythereafter, fluorescence is measured with an excitation wavelength of450 nm and an emission wavelength of 490 nm (Wall J., Schell M., MurphyC., Hrncic R., Stevens F. J., Solomon A. (1999) Thermodynamicinstability of human lambda 6 light chains: correlation withfibrillogenicity, Biochemistry 38(42), 14101-14108).

(C) Detection of Aβ Toxicity

It is known that when Aβ takes a β-sheet structure, Aβ readily formsmass of Aβ fiber and shows toxicity. In the present invention, detectionof Aβ toxicity may be performed using known methods in whichcytotoxicity is measured after addition of Aβ to septal area neurons.Preferable, representative examples of such methods include, but are notlimited to, those described below. For example, Aβ toxicity is examinedusing the amount of LDH in the medium as an indicator in the same manneras in (1) above. Alternatively, in the present invention, Aβ toxicity isexamined by MTT [(dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]assay after addition of Aβ. MTT assay may be performed, for example, byadding MTT (Sigma) to the medium to give a final concentration of 1mg/ml, incubating the medium at 37° C. for 1 hour, removing the medium,solubilizing cells with DMSO, and measuring the absorbance at 550 nm.Alternatively, Aβ toxicity is examined by a cytotoxicity measuringmethod using alamar blue. In this method, alamar blue (WakoPurechemical) is added to the medium at 10% and reacted under 5% CO₂.Four hours after the addition, absorbance was measured with anexcitation wavelength of 530 nm, a fluorescence wavelength of 590 nm andgain 35. Measured results are converted taking the result for mediumalone as 0% and the result for control as 100%.

The Aβ added to cells may be the full-length Aβ or Aβ(1-40) consistingof the N-terminal 40 residues. As long as the sequence takes a β-sheetstructure, Aβ of any length may be used.

Alternatively, the degree of cell death caused by Aβ toxicity may beexamined by immunostaining with anti-MAP2 antibody. Since MAP2 is aneurocyte marker, it is possible to confirm the degree of loss ofneurons induced by Aβ toxicity from the amount of cells expressing MAP2.The degree of cell death may also be examined by a method of measuringcytotoxicity using trypan blue. Briefly, trypan blue is added to cells,and those cells which are not stained with dark blue are counted aslivable cells.

(D) Nerve Protective Agent against Aβ Toxicity and Aβ AggregationInhibitor for Septal Area Neurons

When the siRNA of acetylcholinesterase (ACHE) gene is added to septalarea neurons, intracellular ACHE activity decreases. By measuring theLDH described in (C) above, it is demonstrated that disorders of septalarea neurons induced by Aβ toxicity is ameliorated by treatment withsiRNA.

Further, by measuring the CD spectra described in (B), it isdemonstrated that Aβ aggregation is inhibited by the treatment withsiRNA in the siRNA-added septal area neurons.

From what has been described above, it is clear that ACHE is involved inthe Aβ toxicity and Aβ aggregation in septal area neurons. Therefore, anAβ Aggregation inhibitor comprising a compound which has an effect ofinhibiting the function of acetylcholinesterase and also has an effectof substantially inhibiting the interaction between acetylcholinesteraseand Aβ is included in the present invention.

The term “interaction” means the binding of acetylcholinesterase to Aβ.The expression “substantially inhibiting the interaction betweenacetylcholinesterase and Aβ” means to inhibit the enhancement of Aβaggregation in the presence of acetylcholinesterase. And as long as Aβtoxicity is inhibited, the interaction can be said “substantially”inhibited.

The lowering of the function of acetylcholine may be brought byinhibition of the activity of acetylcholinesterase or by inhibition ofthe expression of acetylcholinesterase gene.

For the inhibition of the activity of ACHE, any compound which inhibitsthe enzyme activity of ACHE may be used as long as it inhibits theinteraction between acetylcholine and Aβ (e.g., donepezil hydrochloriderepresented by the following formula). Alternatively, anti-AChE antibodymay be used.

For the inhibition of the expression of ACHE gene, any nucleic acidinducing RNAi may be used (e.g., siRNA or shRNA) as long as the nucleicacid inhibits the interaction between acetylcholine and Aβ and yetinhibits the expression of ACHE gene. In order to inhibit the expressionof ACHE gene by RNAi, for example, an siRNA (small interfering RNA) orshRNA (short hairpin RNA) for ACHE gene may be designed and synthesized.Then, the siRNA or shRNA may be allowed to act to induce RNAi. siRNA isa short-strand RNA produced intracellularly through the processing ofDicer. On the other hand, shRNA is a small RNA molecule folded into ahairpin structure and has a stem-loop structure in which the sensestrand and the antisense strand are linked with a loop.

Standards for siRNA design are as follows.

-   (a) A region downstream of the initiation codon of a gene encoding    ACHE is selected. Any sequence located downstream of the initiation    codon may be used; any region may be the candidate.

(b) From the selected region, a sequence of consecutive 11-30 bases,preferably 21-25 bases, starting with aa is selected. Specifically,sequences which comprise one of the following nucleotide sequences as atarget sequence and consist of 30 bases or less (e.g., 11-30 bases,preferably 21-25 bases) may be used as siRNA (combination of (i-1) and(i-2); and combination of (ii-1) and (ii-2)). (i-1)5′-AAUGUCAGUGACUCUGUUUTT-3′ (SEQ ID NO: 1) (i-2)5′-AAACAGAGUCACUGACAUUTT-3′ (SEQ ID NO: 2) (ii-1)5′-GCUGCCUCAAGAAAGUAUCTT-3′ (SEQ ID NO: 3) (ii-2)5′-GAUACUUUCUUGAGGCAGCTT-3′ (SEQ ID NO: 4)

In the present invention, the siRNA of ACHE gene is preferably used inthe form of a double-stranded RNA formed by the combination of (i-1) and(i-2) or the combination of (ii-1) and (ii-2).

Standards for shRNA design are as follows.

-   (a) shRNA sequences for ACHE to be used for RNAi are designed in a    region specific to the gene, i.e., from the 5′-terminal region to    300 bases (Elbashir, S. M. et. al. Genes Dev. 15, 188-200 (2001)).-   (b) From a region near 5′ end of individual gene transcripts, mRNA    sequences starting with aa or a are selected and designed as target    sequences. The length of the target region is not particularly    limited. The length is preferably 30 bp or less, more preferably    21-25 bp.

In order to introduce siRNA or shRNA into cultured septal area neurons,various methods may be used. For example, a method in which in vitrosynthesized siRNA or shRNA is linked to a plasmid DNA and this plasmidis introduced into the neurons; or a method of annealing two strands ofRNA may be used.

Further, compounds which are predictable from the structure analysis ofAβ aggregation inhibitory effect-showing siRNA or shRNA and have Aβaggregation inhibitory effect are also included in the presentinvention.

(E) Detection of Aβ Aggregation Inhibitory Effect and Protective Effectagainst Aβ Toxicity by Cholinesterase Inhibitor or siRNA ofCholinesterase Gene

The present invention provides a method of screening for compounds withAβ aggregation inhibitory effect or pharmacologically acceptable saltsthereof. Briefly, it is possible to screen for those compounds whichinhibit Aβ aggregation by adding a test compound (candidate compound) toneurons of the central nervous system in the presence of Aβ and thenmeasuring decrease in CD spectra at 215-260 nm, especially at 215 nm.The judgment whether the test compound has Aβ aggregation inhibitoryeffect or not can be made by comparing the amount of Aβ aggregation inthe presence of the test compound with the amount of Aβ aggregation inthe absence of the test compound. The present invention also provides ascreening kit for compounds with Aβ aggregation inhibitory effect orpharmacologically acceptable salts thereof, to be used for theabove-described method.

The kit of the present invention contains what are necessary formeasuring Aβ aggregation inhibition by the CD spectrum method describedin (B) above. With respect to Aβ necessary for Aβ aggregation andreagents used in the measurement of CD spectra, those used in Examples 6and 7 described later may be used appropriately.

Further, the present invention provides a method of screening forcompounds, or pharmacologically acceptable salts thereof, effective inpreventing and/or treating disorders in neurons of the central nervoussystem induced by Aβ toxicity. Briefly, it is possible to screen forcompounds effective in preventing and/or treating disorders in neuronsof the central nervous system induced by Aβ toxicity by adding Aβ and atest compound to cultured septal area neurons and then detectingcytotoxicity or cell death. The cytotoxicity or cell death can bedetected by measuring the amount of LDH described in (C) above or by MTTassay or the like. The present invention also provide a screening kitfor compounds, or pharmacologically acceptable salts thereof, effectivein preventing and/or treating disorders in neurons of the centralnervous system induced by Aβ toxicity, to be used in the above-describedmethod.

The kit of the present invention contains what are necessary formeasuring disorders in neurons induced by Aβ toxicity using the amountof extracellular LDH described in (C) above as an indicator, or what arenecessary for performing MTT assay or the like. With respect to reagentsnecessary for determining the degree of cytotoxicity induced by Aβtoxicity with LDH, those used in the following Examples 1, 2, 3, 5 and 7may be used appropriately. For example, NADH and pyruvic acid may beused. With respect to reagents necessary for determining the degree ofcytotoxicity induced by Aβ toxicity with MTT, MTT may be used.

These components constituting the kit may be provided separately.Alternatively, they may be provided together as long as there is nohindrance. The kit may contain solutions, or components to prepare suchsolutions may be in a condensed form.

Further, if necessary, the kit may also comprise adjuvants, exclusivecontainers, other necessary accessories, manuals, and the like.

In the present invention, the Aβ aggregation inhibitory effect ofacetylcholinesterase inhibitor (such as donepezil) or the siRNA ofacetylcholinesterase gene can be confirmed by adding theacetylcholinesterase inhibitor before (e.g., 24 hours) adding Aβ andmeasuring the CD spectra described above after (e.g., 48 hours) theaddition of Aβ.

Further, the protective effect against Aβ toxicity ofacetylcholinesterase inhibitor (such as donepezil) or the siRNA ofacetylcholinesterase gene can be confirmed by measuring the amount ofLDH in the medium of cells treated in the same manner as describedabove.

Hereinbelow, the present invention will be described more specificallywith reference to the following Examples which should not be construedas limiting the present invention.

EXAMPLE 1 OGD Test

In this Example, a model is used in which ischemia-like cytotoxicity isinduced in rat primary culture cerebral cortex neurons by giving a loadvia oxygen glucose deprivation. Whether donepezil hydrochloride hasprotective effect on neurons against the ischemia-like disorder or notis examined using that model.

In this Example, primary culture neurons were prepared from the cerebralcortex of rat embryos (17-19-day-old embryos). As a culture medium, DMEM(Gibco BRL) supplemented with 10% fetal calf serum (Gibco BRL), 10%horse serum (Gibco BRL), 5 μg/ml insulin (Sigma), 30 nM sodium selenite(Sigma), 100 μM putrecine (Sigma), 20 nM progesterone (Sigma), 15 nMbiotin (Sigma), 100 units/ml penicillin (Gibco BRL), 100 μg/mlstreptomycin (Gibco BRL), 8 mM glucose and 1 mM pyruvic acid (Sigma) wasused (Scholtz et al., 1988). Cells were cultured at 37° C. under 5% CO₂.After culturing for 7 days or more, cells were subjected to OGDtreatment. Briefly, rat primary culture cerebral cortex neurons wereplaced in a glucose-free buffer (Krebs-Ringer bicarbonate buffer: 5.36mM KCl, 1.26 mM CaCl₂, 0.44 mM KH₂PO₄, 0.49 mM MgCl₂, 0.41 mM MgSO₄, 137mM NaCl, 4.17 mM NaHCO₃, 0.34 mM Na₂HPO₄, 10 mM HEPES (pH 7.4)) and thentransferred into a tightly sealed chamber where nitrogen replacement wascarried out to create a low oxygen environment. Cells after OGDtreatment are transferred from the glucose-free buffer to a cell culturemedium and cultured overnight at 37° C. under 5% CO₂. First, donepezilwas added before and after OGD treatment to examine whether neurocyteprotective effect is observed or not, or whether neurons change or not.Neurocyte protective effects were compared among the following groups:“Pre-12 h” where donepezil was added 12 hours before OGD treatment;“Pre-1 h” where donepezil was added 1 hour before OGD treatment; “Post-1h” where donepezil was added 1 hour after OGD treatment; “Cont” where noOGD treatment was given; and “Vehicle” where no donepezil was addedbefore or after OGD treatment. As an indicator for neurocyte protectioneffect, ratio of LDH release inhibition was used. LDH (lactatedehydrogenase) is an oxidation-reduction enzyme present in cytoplasm andconverts pyruvic acid into lactic acid to thereby reduce the amount ofintracellular NADH (nicotinamide adenine dinucleotide). Therefore, whencells are injured by OGD, LDH flows out from inside of the cells intoextracellular solution. In this solution, LDH is present depending onthe degree of injury of the cells (cell death). The amount of LDHpresent in the solution can be determined by adding pyruvic acid andNADH to the solution and measuring the decreasing ratio of NADH with anabsorption spectrometer. The results are shown in FIG. 1. As clearlyseen from FIG. 1, “Pre-12 h” showed the highest LDH release inhibitoryeffect, and “Pre-1 h” and “Post-1 h” followed in this order. From theseresults, it is understood that donepezil protects neurons effectivelywhen added 12 hours before OGD treatment. The time point of donepeziladdition was fixed at Pre-12 h in any of the subsequent OGD tests.

Subsequently, rat primary culture neurons which had been subjected toOGD treatment in the same manner as in the previous test weremicroscopically observed. The results are shown in FIG. 2. As clearlyseen from FIG. 2, cells in Vehicle (B) do not show the normal state, ascompared with control (A). It is remarkable that cells in Vehicle havebeen damaged by OGD. On the other hand, when donepezil was added beforeOGD treatment, such damage as seen in (B) was not observed and cells areclearly in a state close to that of neurons before OGD treatment (C).Thus, it was demonstrated that neurons were protected by the addition ofdonepezil before OGD treatment.

Subsequently, rat primary culture neurons were subjected to OGDtreatment in the same manner as in the previous test. At that time,various acetylcholinesterase inhibitors (galantamine, tacrine andrivastigmine) and donepezil were added to the cells in variedconcentrations to examine their effect upon LDH release. Theconcentrations used were 0.1, 1.0 and 10 μM for donepezil, tacrine andrivastigmine; and 1.0, 10 and 100 μM for galantamine. The results areshown in FIG. 3. As clearly seen from FIG. 3A, only donepezil inhibitsstatistically significantly the LDH release of neurons in adose-dependent manner. The 50% inhibition concentrations (IC₅₀) ofacetylcholinesterase inhibitors in disrupted suspension of rat brain areshown in Table 1. TABLE 1 AChE Inhibition IC₅₀ (nM) Donepezil 6.7 ± 0.35Galantamine 1200 ± 33   Tacrine 77 ± 1.4  Rivastigmine  4.3 ± 0.087Ogura et al. 2000. Comparison of inhibitory activities of Donepezil andother cholinesterase inhibitors on acetylcholinesterase andbutyrylcholinesterase in vitro. Methods Find Exp Clin Pharmacol. 22,609-613

As is clear from Table 1, the acetylcholinesterase 50% inhibitionconcentration of donepezil is almost equivalent to that of rivastigmine.However, rivastigmine and other acetylcholinesterase inhibitors did notshow any neurocyte protective effect. These results suggest that theneurocyte protective effect of donepezil is based on a mode of actionwhich is different from acetylcholinesterase inhibitory action.

Subsequently, how the neurocyte protective effect of donepezil isaffected when acetylcholine receptor antagonists scopolamine (muscarinereceptor antagonist) and mecamylamine (nicotine receptor antagonist) areadded to cells was examined by the following experiment.

Briefly, in the same manner as in the previous tests, donepezil,scopolamine, mecamylamine, a combination of donepezil and scopolamine,or a combination of donepezil and mecamylamine was added to the culturemedium of neurons 12 hours before OGD treatment. Then, their effects onLDH release induced by OGD treatment were tested. Each of donepezil,scopolamine and mecamylamine was added at the concentration of 10 μM.

The results are shown in FIG. 4. From FIG. 4, it is clear that theneurocyte protective effect of donepezil is not affected by the additionof scopolamine (FIG. 4A) and mecamylamine (FIG. 4B) in any of thesamples of “Cont” (without OGD treatment), “Vehi” (without donepezil)and donepezil-added samples. This indicates that donepezil is protectingneurons regardless of the presence or absence of inhibition byacetylcholine receptors. Therefore, according to this experiment, it isbelieved that donepezil acts neurocyte protectively by a mode of actionother than acetylcholinesterase inhibitory action.

EXAMPLE 2 Excitotoxicity Test

In this Example, the neurocyte protective effect of donepezil againstNMDA toxicity was examined. A model is used in which cytotoxicity isinduced in rat primary culture cerebral cortex neurons by NMDAstimulation. Briefly, 100 μM of NMDA was added to rat primary cultureneurons obtained in the same manner as in Example 1, and the amount ofLDH in the culture medium was measured 9 hours after the addition.

In the present invention, primary culture neurons were prepared from thecerebral cortex of rat embryos (17-19-day-old embryos). As a culturemedium, MEM (Invitrogen) supplemented with glucose (1 g/L),penici./strept. (100 unit/mL) and 10% FCS was used. The medium wasexchanged in every 2 to 3 days. When KA toxicity was examined,Neurobasal Medium (Invitrogen) supplemented with B-27 and glutamine(0.25 mM) was used. Cells were cultured at 37° C. under 5% CO₂. Afterculturing for 7 days or more, 100 μM of NMDA was added to the medium.Then, cells were cultured overnight at 37° C. under 5% CO₂. Donepezilwas added 12 hours before the addition of NMDA stimulation. Nine hoursafter the addition of NMDA, the amount of LDH in the culture medium wasmeasured and used as an indicator for nerve protective effect.

The results of this Example are shown in FIG. 5. From FIG. 5, it isunderstood that donepezil inhibits the LDH release of neurons in a dosedependent manner in a range from 0.1 μM to 10 μM. According to thisexperiment, it has become clear that donepezil shows neurocyteprotective effect against NMDA excitotoxicity in a dose dependentmanner.

EXAMPLE 3 Excitotoxicity Test

It is said that kainic acid enhances the death of neurons induced byβ-amyloid (Aβ) which is said one of the causative factors of Alzheimer'sdisease. In this Example, a model is used in which cytotoxicity isinduced in rat primary culture cerebral cortex neurons by addition ofkainic acid.

In the present invention, primary culture neurons were prepared from thecerebral cortex of rat embryos (17-19-day-old embryos). Cells werecultured at 37° C. under 5% CO₂. After culturing for 7 days or more,NMDA or kainic acid was added to the medium. Then, cells were culturedovernight at 37° C. under 5% CO₂. Donepezil was added 24 hours beforethe addition of kainic acid stimulation. Twenty-four hours after theaddition of kainic acid, the amount of LDH in the culture medium wasmeasured and used as an indicator for nerve protective effect.

The results of this Example are shown in FIG. 6. From FIG. 6, it isunderstood that donepezil inhibits the LDH release of neurons in a dosedependent manner in a range from 0.1 μM to 1 μM. According to thisexperiment, it has become clear that donepezil shows neurocyteprotective effect against kainic acid excitotoxicity in a dose dependentmanner.

The reagents and statistical analysis methods used in the followingExamples 4 to 7 are as described below. Aβ (human, 1-40) was purchasedfrom Peptide Institute, Inc. (Osaka, Japan). Trypsin solution,penicillin, streptomycin, Dulbecco's modified Eagle's medium (DMEM) andHEPES were purchased from Life Technologies Inc. (Grand Island, N.Y.).Insulin, sodium selenite, putrescine, DNase I, mecamylamine andscopolamine were purchased from Sigma Chemical Co. (St. Louis, Mo.).Fetal calf serum and heat-inactivated horse serum were purchased fromNichirei Co. (Tokyo, Japan). ChAT and microtubule associated protein-2(MAP2) were purchased from Chemicon International Inc. (Temecula,Calif.). Alexa Fluor 546 was purchased from Molecular Probes (Eugene,Oreg.). Vecstain Elite ABC kit and diaminobenzidine (DAB) kit werepurchased from Vector Laboratories, Inc. (Burlingame, Calif.).Lipofectamine 2000 was purchased from Invitrogen Co. (Carlsbad, Calif.).siRNA was synthesized by Japan BioService (Saitama, Japan).

Statistical data are shown as mean±standard error. For comparative test,Welch's test was used. p<0.05 was regarded as significantly different.The statistical analysis software used was SAS 8.1 (SAS Institute JapanLtd., Tokyo, Japan).

EXAMPLE 4 Culture of Septal Area Neurons and Confirmation of Their BeingCholinergic Nerves

Septal area neurons were prepared from embryos (18-day-old embryos) ofWistar rats (Charles River Japan). The septal region (including septalarea and basal forebrain) was cut out from embryonic brain and incubatedin Ca²⁺/Mg²⁺-free Hanks' balanced salt solution containing 0.25% trypsinand 0.2 mg/ml DNase I at 37° C. for 15 minutes. To the resultant cells,10% fetal calf serum and 10% horse serum-added DMEM (containing 5 μg/mlinsulin, 30 nmol/L sodium selenite, 100 μmol/L putrecine, 20 nmol/Lprogesterone, 15 nmol/L biotin, 100 units/ml penicillin, 100 μg/mlstreptomycin and 1 mmol/L sodium pyruvate) was added to deactivatetrypsin, and then cells were washed twice. Cells were plated on 96-wellpoly-D-lysine-coated culture plates at a concentration of 1.2×10⁵cells/well. The plated cells were cultured in a CO₂ incubator (5% CO₂,37° C.). After one day, ⅔ of the medium was exchanged for theabove-described DMEM containing 5% fetal calf serum. After three days, ⅔of the medium was exchanged again for the above-described DMEMcontaining 5% fetal calf serum. After 6 days, the entire medium wasexchanged for the above-described DMEM (this time, Gln is not contained)not containing serum.

The thus cultured septal area neurons were immunostained withanti-choline acetyltransferase (ChAT) antibody. First, the culturedseptal area neurons were fixed in a neutral formalin solution for 60minutes. After 30 minute incubation with 5% goat serum, the cells wereincubated with a 500-fold dilution of ChAT antibody in the presence of1% goat serum for one day. Subsequently, the cells were incubated with a500-fold dilution of goat anti-rabbit IgG and HRP-labeled avidin-biotincomplex for one hour, followed by staining with DAB as the substrate forperoxidase.

The results of the immunostaining of cultured rat septal area neuronswith anti-ChAT antibody are shown in FIG. 7. The scale bar in FIG. 7represents 0.1 mm. The immunostaining revealed that a great number ofthe cultured rat septal area neurons have choline acetyltransferase(ChAT) (an acetylcholine synthesis enzyme). This means that the culturedrat septal area neurons are cholinergic nerves. Thus, it could beconfirmed that the cells are cholinergic nerves.

It has been reported that the decrease of choline acetyltransferase inthe brains of Alzheimer patients correlates to the severity of cognitivedisorders (Perry E. K., Tomlinson B. E., Blessed G., Bergman K., GibsonP. H. and Perry R. H. (1978) Correlation of cholinergic abnormalitieswith senile plaques and mental test scores in senile dementia. Br. Med.J. 2, 1457-1459). Therefore, it can be said that the inhibition ofdisorders in septal area neurons leads to inhibition of the lowering ofcognitive function in Alzheimer patients.

EXAMPLE 5 Protective Effect of Donepezil against Aβ(1-40) Toxicity andCell Death Experiment

As an indicator for cytotoxicity, LDH (lactate dehydrogenase) wasmeasured. As cells, septal area neurons cultured for 7 days according tothe culture method in Example 4 were used. Briefly, 15 μmol/L ofAβ(1-40) was added to the medium of septal area neurons, and the cellswere cultured in a CO₂ incubator (5% CO₂, 37° C.). Donepezil (0.01, 0.1,1, 10 μmol/L), mecamylamine (10 μmol/L) and scopolamine (10 μmol/L) wereadded 24 hours before the addition of Aβ(1-40). Forty-eight hours afterthe addition of Aβ(1-40), lactate dehydrogenase (LDH) activity in themedium was measured. At the same time, cells were solubilized withphosphate buffer (pH 7.4) containing 0.5% Triton X-100, followed bymeasurement of LDH in cells. Out of the total LDH (present in the cellsand in the medium), the amount of LDH which leaked out into the mediumwas determined and expressed in % (Koh J. Y. and Choi D. W. (1987)Quantitative determination of glutamate mediated cortical neuronalinjury in cell culture by lactate dehydrogenase efflux assay. J.Neurosci. Meth. 20, 83-90). The data were analyzed by analysistechniques of ANOVA and Welch's test.

The results are shown in FIG. 8. In FIG. 8, open columns represent cellsto which no Aβ was added; and gray columns represent cells to which Aβwas added. Individual LDH values were determined as mean±S.E.M.(n=23-25) and summarized in a graph. In FIG. 8, mark “*” representsp<0.05; mark “**” represents p<0.01; and mark “***” represents p<0.001(“control cells” was used as control).

The cultured septal area neurons were susceptible to disorders by theaddition of Aβ(1-40) (see “control cells” in FIG. 8). Donepezil showedsignificant protective effect from 0.1 μmol/L against the toxicity whenAβ(1-40) was added for 48 hours (FIG. 8). The inhibitory effect ofdonepezil against Aβ(1-40) toxicity was not inhibited by acetylcholinereceptor antagonists mecamylamine and scopolamine (FIG. 8).

Subsequently, the cultured rat septal area neurons treated with Aβ(1-40)in the same manner as described above were immunostained with anti-MAP2antibody. MAP2 is used as a neurocyte marker (Herzog W. and Weber K.(1978) Fractionation of brain microtubule-associated proteins. Isolationof two different proteins which stimulate tubulin polymerization invitro. Eur J Biochem. 92(1), 1-8). MAP2 staining was performed based onthe immunostaining method described in Example 4 above. Briefly, 48hours after the addition of 15 μmol/L of Aβ(1-40) to the cells, thecells were fixed and immunostained. Donepezil was added in advance 24hours before the addition of A(1-40). After fixation, the cells wereincubated with a 300-fold dilution of anti-MAP2 antibody for one day,followed by detection using Alexa Fluor546 as a fluorescent substrate.

The results are shown in FIG. 9. The scale bar represents 0.1 mm. PanelA shows control cells to which Aβ(1-40) was not added. Panel B showscells to which Aβ(1-40) was added. Panels C, D and E show cells to whicha combination of Aβ(1-40) and donepezil (0.1, 1 and 10 μmol/L) wasadded. The photograph at the left in each panel is a bright fieldphotograph by Hoffman modulation. The photograph at the right in eachpanel is a photograph of fluorescence. When Aβ(1-40) was added for 48hours, the number of neurons stained with MAP2 decreased (FIG. 9B). Itwas recognized that donepezil inhibits the disorders in MAP2-stainedcells induced by Aβ(1-40) (FIG. 9).

EXAMPLE 6 Effect of Donepezil on Aβ(140) Aggregation

In order to examine the structural state of Aβ(1-40) in culture medium,CD spectra were measured. It is known that when Aβ takes a β-sheetstructure, Aβ readily forms mass of Aβ fiber and shows toxicity (HowlettD. R., Jennings K. H., Lee D. C., Clark M. S., Brown F., Wetzel R., WoodS. J., Camilleri P. and Roberts G. W. (1995) Aggregation state andneurotoxic properties of Alzheimer beta-amyloid peptide.Neurodegeneration. 4, 23-32). It is also known that CD (circulardichroism) spectra at 205-225 nm decrease when Aβ(1-49) forms an α-helixstructure or a β-sheet structure (Sreerama N., Venyaminov S. Y. andWoody R. W. (2000) Estimation of peptide secondary structure fromcircular dichroism spectra: inclusion of denatured peptides with nativepeptides in the analysis. Anal. Biochem. 287, 243-251; Bokvist M.,Lindstrom F., Watts A. and Grobner G. (2004) Two types of Alzheimer'sbeta-amyloid (1-40) peptide membrane interactions: aggregationpreventing transmembrane anchoring versus accelerated surface fibrilformation. J. Mol. Biol. 335, 1039-49). In particular, it is known thatthe formation of a β-sheet structure decreases CD spectra around 215 nm.Then, CD was measured as an indicator for Aβ aggregation.

Briefly, 15 μmol/L of Aβ(1-40) was added to cultured septal areaneurons. Forty-eight hours after the addition, medium samples weretaken, followed by measurement of CD spectra at 215-260 nm. A JASCOspectrometer model J-720WI (JASCO Corporation) was used for themeasurement.

The results are shown in FIG. 10. Panel A shows cells to which donepezilwas not added. Panel B shows cells to which donepezil (10 μM) was added.Solid line represents cells to which Aβ was not added. Dotted linerepresents cells to which Aβ was added. Panels A2 and B2 show graphsobtained by subtracting the values of the solid lines from the values ofthe dotted lines in graphs A and B, respectively. The decrease of CDspectra at 215-225 nm was inhibited by the addition of 10 μmol/L ofdonepezil. Therefore, according to this Example, it was demonstratedthat donepezil inhibits the formation of α-helix structure or β-sheetstructure by Aβ.

EXAMPLE 7 Effect of siRNA on Intracellular Acetylcholine Activity andIts Protective Effect against Aβ(1-40) Toxicity

(1) siRNA Treatment

The following two sets of oligonucleotides [(i) and (ii)] were used asthe double-stranded siRNAs of acetyl cholinesterase. (i);5′-AAUGUCAGUGACUCUGUUUTT-3′ (SEQ ID NO: 1) 5′-AAACAGAGUCACUGACAUUTT-3′(SEQ ID NO: 2) and (ii); 5′-GCUGCCUCAAGAAAGUAUCTT-3′ (SEQ ID NO: 3)5′-GAUACUUUCUUGAGGCAGCTT-3′ (SEQ ID NO: 4)

On day 6 and 7 of culture, 1 μmol/L of siRNA (i) and siRNA (ii) aboveand lipofectamine 2000 (0.4 μL/mL) were added to the medium of septalarea neurons. On day 9 of culture, cells were solubilized andintracellular acetylcholinesterase activity (ACHE activity) wasmeasured.

(2) Measurement of Acetylcholinesterase Activity

Measurement of acetylcholinesterasesterase activity was performedaccording to the method of Ellman et al. (Ellman G. L., Courtney K. D.,Anders V. J., Feather-Stone R. M. (1961) A new and rapid colorimetricdetermination of acetylcholinesterase activity. Biochem. Pharmacol. 7,88-95). Briefly, cells were solubilized in 0.5% Triton X-100-containingphosphate buffer (pH 8.0). The resultant solubilized cell solution wascentrifuged, and the supernatant was diluted 3-fold with phosphatebuffer (pH 8.0). To this diluted supernatant,5,5′-dithiobis(2-nitrobenzoic acid) (0.33 mmol/L) and acetylthiocholineiodide (AthCh) (0.5 mmol/L) were added and agitated. Then, increase inabsorbance at 412 nm was measured (Molecular Devices, SpectraMax190EXT). The data were analyzed by ANOVA and Welch's test.

The results are shown in FIG. 11. Individual values were obtained asmean±S.E.M. (n=1) and summarized in a graph (n=11). In FIG. 11, mark“**” represents p<0.01 and mark “***” represents p<0.001, relative tothe cells which were not treated with siRNA.

As a result of examination of two siRNAs (i) and (ii), it was recognizedthat both siRNAs have AChE activity inhibitory effect (FIG. 11).

(3) Protective Effect of siRNA against Aβ(1-40) Toxicity

LDH (lactate dehydrogenase) was measured as an indicator forcytotoxicity. Briefly, 15 μmol/L of Aβ(1-40) was added to culturedseptal area neurons on day 7 of culture and incubated for 48 hours.siRNA (1 μmol/L) was added 24 hours before and immediately before theaddition of Aβ(1-40). Open columns represent cells to which Aβ(1-40) wasnot added. Gray columns represent cells to which Aβ(1-40) was added. Thedata were analyzed by ANOVA and Welch's test.

The results are shown in FIG. 12. Individual values were obtained asmean±S.E.M. and summarized in a graph (n=7). In FIG. 12, mark “***”represents p<0.001, relative to the cells which were not treated withsiRNA.

As a result of examination of two siRNAs (i) and (ii), it was found thatboth siRNAs inhibit Aβ toxicity (FIG. 12).

In parallel with the measurement of LDH, Aβ aggregation was analyzedwith changes in CD spectra at 215-260 nm.

The results are shown in FIG. 13. Panel A shows cells to which RNAi wasadded. Solid line represents cells to which Aβ was not added. Dottedline represents cells to which Aβ was added. Panel B shows the valuesobtained by subtracting the values of dotted line from the values ofsolid line. Decrease in CD spectra at 215-225 nm was inhibited by theaddition of siRNA (FIG. 13).

INDUSTRIAL APPLICABILITY

The neurocyte protective agent of the present invention works in aneurocyte protective manner against ischemia-like cytotoxicity andinjury induced by excitotoxicity. Therefore, with the neurocyteprotective agent of the present invention, it is possible to prevent orinhibit the ischemic death of neurons induced by cerebral infarction,cerebral thrombosis, or the like and the death of neurons induced byexcitotoxicity.

Sequence Listing Free Text

-   SEQ ID NO: 1: siRNA-   SEQ ID NO: 2: siRNA-   SEQ ID NO: 3: siRNA-   SEQ ID NO: 4: siRNA

1. A protective agent for neurons of the central nervous system,comprising any one of the compounds shown in the following (i) to (vii):(i) 1-benzyl-4-[(5,6-dimethoxy-1-indanone)-2-yl]methylpiperidinerepresented by the following chemical formula or a pharmacologicallyacceptable salt thereof:

(ii) a cyclic amine derivative represented by the following generalformula (I) or a pharmacologically acceptable salt thereof:

where J is a monovalent or divalent group selected from the groupsrepresented by the following formulas:

where S is a lower alkyl group with 1 to 6 carbon atoms, a lower alkoxygroup with 1 to 6 carbon groups, a halogen atom or a hydroxyl group; tis 0 or an integer from 1 to 4; (S)_(t) may form a methylenedioxy orethylenedioxy group between adjacent carbon atoms on the phenyl ringlinked; Y in formula (l) is a hydrogen atom or a lower alkyl group with1 to 6 carbon atoms; V in formula (k) is a hydrogen atom or a loweralkoxy group with 1 to 6 carbon atoms; W¹ and W² in formula (n)independently represent, similarly or differently, a hydrogen atom, alower alkyl group with 1 to 6 carbon atoms, or a lower alkoxy group with1 to 6 carbon atoms; W³ in formula (n) is a hydrogen atom or a loweralkyl group with 1 to 6 carbon atoms; phenyl ring A in formulas (a) to(e), (g), (j), (l) and (q) may be substituted with an alkyl group with 1to 6 carbon atoms or a alkoxy group with 1 to 6 carbon atoms; B is agroup represented by a formula —(CHR²)_(n)— (where n is 0 or an integerfrom 1 to 10; R² is each independently a hydrogen atom or a methylgroup), a group represented by a formula ═(CH—CH═CH)_(b)— (where b is aninteger from 1 to 3), a group represented by a formula ═CH—(CH₂)_(c)—(where c is 0 or an integer from 1 to 9), or a group represented by aformula ═(CH—CH)_(d)═ (where d is 0 or an integer from 1 to 5); K is aphenylalkyl group that may have, as a substituent, an alkyl group with 1to 6 carbon atoms which may be halogenated, an alkoxy group with 1 to 6carbon atoms, a nitro group, a halogen atom, a carboxyl group, abenzyloxy group, an alkoxycarbonyl group with 1 to 6 carbon atoms, anamino group, a monoalkylamino group with 1 to 6 carbon atoms, adialkylamino group with 1 to 6 carbon atoms, a carbamoyl group, anacylamino group with 1 to 6 carbon atoms, a cyclohexyloxycarbonyl group,an alkylaminocarbonyl group with 1 to 6 carbon atoms, analkylcarbonyloxy group with 1 to 6 carbon atoms, a hydroxyl group, aformyl group or an alkoxy (with 1 to 6 carbon atoms)-alkyl (with 1 to 6carbon atoms) group; and

represents a single bond or a double bond; (iii) a cyclic aminederivative selected from the compounds represented by the followingformulas or a pharmacologically acceptable salt thereof:

(iv) a cyclic amine derivative selected from the compounds representedby the following formulas or a pharmacologically acceptable saltthereof:

(v) a cyclic amine derivative represented by the following generalformula (I-1) or a pharmacologically acceptable salt thereof:

where J^(I-1) is a lower alkyl group with 1 to 6 carbon atoms(hereinafter, just referred to as “lower alkyl group”); a cyclohexylgroup; a phenyl, pyridyl or pyrazyl group which may have, as asubstituent, a lower alkyl group, a lower alkoxy group with 1 to 6carbon atoms (hereinafter, just referred to as “lower alkoxy group”), anitro group, a halogen, a carboxyl group, a lower alkoxycarbonyl group,an amino group, a mono-lower alkylamino group, a di-lower alkylaminogroup, a carbamoyl group, an acylamino group derived from aliphaticsaturated monocarboxylic acid with 1 to 6 carbon atoms, acyclohexyloxycarbonyl group, a lower alkylaminocarbonyl group, a loweralkylcarbonyloxy group, a halogenated lower alkyl group, a hydroxylgroup, a formyl group or a lower-alkoxy-lower-alkyl group; a grouprepresented by a formula

where G is a group represented by a formula

a group represented by a formula

a group represented by a formula —O—, a group represented by a formula

a group represented by a formula —CH₂—O—, a group represented by aformula —CH₂—SO₂—, a group represented by a formula

or a group represented by a formula

E is a carbon atom or a nitrogen atom; a quinolyl group; a quinoxalylgroup; a furyl group or a group represented by a formula R¹—CH═CH—(where R¹ is a hydrogen atom or a lower alkoxycarbonyl group); B is agroup represented by a formula —(CH₂)_(n)— a group represented by aformula —NR²—(CH₂)_(n)— (where R² is a hydrogen atom, a lower alkylgroup, a phenyl group or a lower alkylsulfonyl group), a grouprepresented by a formula —CONR³—(CH₂)_(n)— (where R³ is a hydrogen atom,a lower alkyl group, a phenyl, benzyl or pyridyl group which may have,as a substituent, a lower alkyl group, a lower alkoxy group, a halogenor a hydroxyl group), a group represented by a formula—NH—CO—(CH₂)_(n)—, a group represented by a formula—CH₂—CO—NH—(CH₂)_(n)—, a group represented by a formula—CO—CH₂—CH(OH)—CH₂—, a group represented by a formula —CO—(CH₂)_(n)—, agroup represented by a formula —C(OH)—(CH₂)_(n)— or a group representedby a formula —CO—CH═CH—CH₂—; and n in the above formulas is 0 or aninteger from 1 to 10; T¹ is a carbon atom; K is a phenylalkyl group(where the alkyl has 1 to 2 carbon atoms) in which the phenyl may have,as a substituent, a lower alkyl group, a lower alkoxy group, a nitrogroup, a halogen, a carboxyl group, a lower alkoxycarbonyl group, anamino group, a mono-lower alkylamino group, a di-lower alkylamino group,a carbamoyl group, an acylamino group derived from aliphatic saturatedmonocarboxylic acid with 1 to 6 carbon atoms, a cyclohexyloxycarbonylgroup, a lower alkylaminocarbonyl group, a lower alkylcarbonyloxy group,a halogenated lower alkyl group, a hydroxyl group, a formyl group or alower-alkoxy-lower-alkyl group; a cinnamyl group; a lower alkyl group; apyridyl methyl group; a cycloalkyl (with 3 to 6 carbon atoms)-alkylgroup; an adamantanemethyl group; a furfuryl group; a cycloalkyl groupwith 3 to 6 carbon atoms; or an acyl group; and q is 1 or 2; (vi) acyclic amine derivative represented by the following general formula(I-2) or a pharmacologically acceptable salt thereof:

where J¹⁻² is an indanonyl group which may have, as a substituent, alower alkyl group with 1 to 6 carbon atoms or a lower alkoxy group with1 to 6 carbon atoms; T² is a nitrogen atom; B, K and q are the same asdefined above; (vii) a cyclic amine derivative selected from thecompounds represented by the following formulas or a pharmacologicallyacceptable salt thereof:


2. The protective agent according to claim 1, wherein the salt is ahydrochloride salt.
 3. A prophylactic and/or therapeutic agent fordisorders in neurons of the central nervous system, comprising any oneof the compounds shown in the following (i) to (vii): (i)1-benzyl-4-[(5,6-dimethoxy-1-indanone)-2-yl]methylpiperidine representedby the following chemical formula or a pharmacologically acceptable saltthereof:

(ii) a cyclic amine derivative represented by the following generalformula (I) or a pharmacologically acceptable salt thereof:

where J is a monovalent or divalent group selected from the groupsrepresented by the following formulas:

where S is a lower alkyl group with 1 to 6 carbon atoms, a lower alkoxygroup with 1 to 6 carbon groups, a halogen atom or a hydroxyl group; tis 0 or an integer from 1 to 4; (S)_(t) may form a methylenedioxy orethylenedioxy group between adjacent carbon atoms on the phenyl ringlinked; Y in formula (l) is a hydrogen atom or a lower alkyl group with1 to 6 carbon atoms; V in formula (k) is a hydrogen atom or a loweralkoxy group with 1 to 6 carbon atoms; W¹ and W² in formula (n)independently represent, similarly or differently, a hydrogen atom, alower alkyl group with 1 to 6 carbon atoms, or a lower alkoxy group with1 to 6 carbon atoms; W³ in formula (n) is a hydrogen atom or a loweralkyl group with 1 to 6 carbon atoms; phenyl ring A in formulas (a) to(e), (g), (j), (l) and (q) may be substituted with an alkyl group with 1to 6 carbon atoms or a alkoxy group with 1 to 6 carbon atoms; B is agroup represented by a formula —(CHR²)_(n)— (where n is 0 or an integerfrom 1 to 10; R² is each independently a hydrogen atom or a methylgroup), a group represented by a formula ═(CH—CH═CH)_(b)— (where b is aninteger from 1 to 3), a group represented by a formula ═CH—(CH₂)_(c)—(where c is 0 or an integer from 1 to 9), or a group represented by aformula ═(CH—CH)_(d)═ (where d is 0 or an integer from 1 to 5); K is aphenylalkyl group that may have, as a substituent, an alkyl group with 1to 6 carbon atoms which may be halogenated, an alkoxy group with 1 to 6carbon atoms, a nitro group, a halogen atom, a carboxyl group, abenzyloxy group, an alkoxycarbonyl group with 1 to 6 carbon atoms, anamino group, a monoalkylamino group with 1 to 6 carbon atoms, adialkylamino group with 1 to 6 carbon atoms, a carbamoyl group, anacylamino group with 1 to 6 carbon atoms, a cyclohexyloxycarbonyl group,an alkylaminocarbonyl group with 1 to 6 carbon atoms, analkylcarbonyloxy group with 1 to 6 carbon atoms, a hydroxyl group, aformyl group or an alkoxy (with 1 to 6 carbon atoms)-alkyl (with 1 to 6carbon atoms) group; and

represents a single bond or a double bond; (iii) a cyclic aminederivative selected from the compounds represented by the followingformulas or a pharmacologically acceptable salt thereof:

(iv) a cyclic amine derivative selected from the compounds representedby the following formulas or a pharmacologically acceptable saltthereof:

(v) a cyclic amine derivative represented by the following generalformula (I-1) or a pharmacologically acceptable salt thereof:

where J¹⁻¹ is a lower alkyl group with 1 to 6 carbon atoms (hereinafter,just referred to as “lower alkyl group”); a cyclohexyl group; a phenyl,pyridyl or pyrazyl group which may have, as a substituent, a lower alkylgroup, a lower alkoxy group with 1 to 6 carbon atoms (hereinafter, justreferred to as “lower alkoxy group”), a nitro group, a halogen, acarboxyl group, a lower alkoxycarbonyl group, an amino group, amono-lower alkylamino group, a di-lower alkylamino group, a carbamoylgroup, an acylamino group derived from aliphatic saturatedmonocarboxylic acid with 1 to 6 carbon atoms, a cyclohexyloxycarbonylgroup, a lower alkylaminocarbonyl group, a lower alkylcarbonyloxy group,a halogenated lower alkyl group, a hydroxyl group, a formyl group or alower-alkoxy-lower-alkyl group; a group represented by a formula

where G is a group represented by a formula

a group represented by a formula

a group represented by a formula —O—, a group represented by a formula

a group represented by a formula —CH₂—O—, a group represented by aformula —CH₂—SO₂—, a group represented by a formula

or a group represented by a formula

E is a carbon atom or a nitrogen atom; a quinolyl group; a quinoxalylgroup; a furyl group or a group represented by a formula R¹—CH═CH—(where R¹ is a hydrogen atom or a lower alkoxycarbonyl group); B is agroup represented by a formula —(CH₂)_(n)—, a group represented by aformula —NR²—(CH₂)_(n)— (where R² is a hydrogen atom, a lower alkylgroup, a phenyl group or a lower alkylsulfonyl group), a grouprepresented by a formula —CONR³—(CH₂)_(n)— (where R³ is a hydrogen atom,a lower alkyl group, a phenyl, benzyl or pyridyl group which may have,as a substituent, a lower alkyl group, a lower alkoxy group, a halogenor a hydroxyl group), a group represented by a formula—NH—CO—(CH₂)_(n)—, a group represented by a formula—CH₂—CO—NH—(CH₂)_(n)—, a group represented by a formula—CO—CH₂—CH(OH)—CH₂—, a group represented by a formula —CO—(CH₂)_(n)—, agroup represented by a formula —C(OH)—(CH₂)_(n)— or a group representedby a formula —CO—CH═CH—CH₂—; and n in the above formulas is 0 or aninteger from 1 to 10; T¹ is a carbon atom; K is a phenylalkyl group(where the alkyl has 1 to 2 carbon atoms) in which the phenyl may have,as a substituent, a lower alkyl group, a lower alkoxy group, a nitrogroup, a halogen, a carboxyl group, a lower alkoxycarbonyl group, anamino group, a mono-lower alkylamino group, a di-lower alkylamino group,a carbamoyl group, an acylamino group derived from aliphatic saturatedmonocarboxylic acid with 1 to 6 carbon atoms, a cyclohexyloxycarbonylgroup, a lower alkylaminocarbonyl group, a lower alkylcarbonyloxy group,a halogenated lower alkyl group, a hydroxyl group, a formyl group or alower-alkoxy-lower-alkyl group; a cinnamyl group; a lower alkyl group; apyridyl methyl group; a cycloalkyl (with 3 to 6 carbon atoms)-alkylgroup; an adamantanemethyl group; a furfuryl group; a cycloalkyl groupwith 3 to 6 carbon atoms; or an acyl group; and q is 1 or 2; (vi) acyclic amine derivative represented by the following general formula(I-2) or a pharmacologically acceptable salt thereof:

where J¹⁻² is an indanonyl group which may have, as a substituent, alower alkyl group with 1 to 6 carbon atoms or a lower alkoxy group with1 to 6 carbon atoms; T² is a nitrogen atom; B, K and q are the same asdefined above; (vii) a cyclic amine derivative selected from thecompounds represented by the following formulas or a pharmacologicallyacceptable salt thereof:


4. The prophylactic and/or therapeutic agent according to claim 3,wherein the salt is a hydrochloride salt.
 5. The prophylactic and/ortherapeutic agent according to claim 3, wherein the neurocyte disorderis induced by cerebral ischemia, excitotoxicity or Aβ toxicity.
 6. Theprophylactic and/or therapeutic agent according to claim 3, wherein theneurocyte disorder is induced by cerebral ischemia or excitotoxicityassociated with any one of cerebral apoplexy, cerebral infarction orcerebral embolism.
 7. The prophylactic and/or therapeutic agentaccording to claim 6, wherein the excitotoxicity is by NMDA or kainicacid.
 8. The prophylactic and/or therapeutic agent according to claim 3,wherein the neurocyte disorder is induced by Aβ toxicity associated withAlzheimer's disease or Down's syndrome.
 9. The agent according to claim1, wherein the neurons are brain-derived, mature neurons.
 10. The agentaccording to claim 9, wherein the neurons are derived from any one ofcerebral cortex, septal area or hippocampus.
 11. The agent according toclaim 9, wherein the neurons are primary culture cells.
 12. A prognosisimproving agent for any disease selected from cerebral apoplexy,cerebral infarction or cerebral embolism, comprising the protectiveagent according to claim 1 or 2 or the prophylactic and/or therapeuticagent according to claim 3 or
 4. 13. A method of protecting neurons ofthe central nervous system, comprising administering to a patient aneffective amount of the protective agent according to claim
 1. 14. Amethod of preventing and/or treating disorders in neurons of the centralnervous system, comprising administering to a patient an effectiveamount of the prophylactic and/or therapeutic agent according to claim3.
 15. The method according to claim 14, wherein the disorder in neuronsof the central nervous system is induced by cerebral ischemia,excitotoxicity or Aβ toxicity.
 16. The method according to claim 15,wherein the excitotoxicity is induced by NMDA or kainic acid.
 17. Themethod according to claim 14, wherein the disorder in neurons of thecentral nervous system is induced by cerebral ischemia or excitotoxicityassociated with any one of cerebral apoplexy, cerebral infarction orcerebral embolism.
 18. The method according to claim 14, wherein thedisorder in neurons of the central nervous system is induced by Aβtoxicity associated with Alzheimer's disease or Down's syndrome.
 19. Amethod of improving the prognosis of any one of cerebral apoplexy,cerebral infarction or cerebral embolism, comprising administering to apatient an effective amount of the prognosis improving agent accordingto claim
 12. 20. (canceled)
 21. A method of screening for a compoundwith Aβ aggregation inhibitory effect or a pharmacologically acceptablesalt thereof, comprising contacting cholinergic neurons of the centralnervous system with a candidate compound in the presence of Aβ anddetecting or measuring the amount of Aβ aggregation.
 22. The methodaccording to claim 21, wherein the results of detection or measurementof the amount of Aβ aggregation are compared with the amount of Aβaggregation in the absence of the candidate compound to thereby judgewhether or not the candidate compound has Aβ aggregation inhibitoryeffect.
 23. A screening kit for a compound with Aβ aggregationinhibitory effect or a pharmacologically acceptable salt thereof, whichis for use in the method according to claim
 21. 24. A method forscreening for a compound, or a pharmacologically acceptable saltthereof, effective for preventing and/or treating disorders in neuronsof the central nervous system induced by Aβ toxicity, comprisingcontacting cholinergic neurons of the central nervous system with acandidate compound in the presence of Aβ and detecting cytotoxicity orcell death.
 25. The method according to claim 24, wherein the results ofdetection of cytotoxicity or cell death are compared with the extent ofcytotoxicity or cell death in the absence of the candidate compound tothereby judge whether or not the candidate compound has cell protectiveeffect against Aβ toxicity.
 26. The method according to claim 24,wherein the detection of cytotoxicity or cell death is performed bymeasuring the concentration of lactate dehydrogenase or by MTT assay.27. A screening kit for a compound, or a pharmacologically acceptablesalt thereof, effective for preventing and/or treating disorders inneurons of the central nervous system induced by Aβ toxicity, which isfor use in the method according to claim 24.